Modified proteases

ABSTRACT

This invention relates to modified polynucleotides encoding modified proteases, and methods for altering the production of proteases in microorganisms. In particular, the present invention relates to methods for altering the expression of proteases in microorganisms, such as  Bacillus  species. The invention discloses modified polynucleotides, vectors, modified polypeptides, and processes for enhancing the production of proteases.

This application is a divisional of U.S. patent application Ser. No.12/047,157, filed Mar. 12, 2008, and claims priority to U.S. provisionalapplication Ser. No. 60/906,734, filed Mar. 12, 2007, both of which areherein incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing, which has been submittedvia EFS, in compliance with 37 C.F.R. §1.52(e), and is herebyincorporated by reference in its entirety. The sequence listing textfile submitted via EFS contains the file“30963-US-DIV_SequenceListing.txt” created on Jun. 12, 2014, which is94,530 bytes in size.

FIELD OF THE INVENTION

This invention provides modified polynucleotides encoding modifiedproteases, and methods for altering the production of proteases inmicroorganisms. In particular, the present invention relates to methodsfor altering the expression of proteases in microorganisms, such asBacillus species. The invention discloses modified polynucleotides,vectors, modified polypeptides, and processes for enhancing theproduction of proteases.

BACKGROUND

Microorganisms, such as the Gram-positive microorganism that are membersof the genus Bacillus, have been used for large-scale industrialfermentation due, in part, to their ability to secrete theirfermentation products into their culture media. Secreted proteins areexported across a cell membrane and a cell wall, and then aresubsequently released into the external media. Secretion of polypeptidesinto periplasmic space or into their culture media is subject to avariety of parameters, which need to be carefully considered inindustrial fermentations.

Indeed, secretion of heterologous polypeptides is a widely usedtechnique in industry. Typically, cells are transformed with a nucleicacid encoding a heterologous polypeptide of interest to be expressed andsecreted to produce large quantities of desired polypeptides. Thistechnique has been used to produce large quantities of polypeptides.Expression and secretion of desired polypeptides has been controlledthrough genetic manipulation of the polynucleotides that encode thedesired proteins. Despite various advances in protein productionmethods, there remains a need in the art to provide efficient methodsfor extracellular protein secretion.

SUMMARY OF THE INVENTION

This invention relates to modified polynucleotides encoding modifiedproteases, and methods for altering the production of proteases inmicroorganisms. In particular, the present invention relates to methodsfor altering the production of proteases in microorganisms, such asBacillus species. The invention discloses modified polynucleotides,vectors, modified polypeptides, and processes for enhancing theproduction of proteases.

The present invention relates to polynucleotides, polypeptides and cellsthat have been genetically manipulated to enhance the production ofmodified proteins. In particular, the present invention relates toGram-positive microorganisms having exogenous nucleic acid sequencesintroduced therein and methods for producing proteins in such hostcells, such as members of the genus Bacillus. More specifically, thepresent invention relates to the production of proteases and to cellsthat have been genetically manipulated to have an altered capacity toproduce the expressed proteins. In particular, the present inventionprovides for the enhanced production of proteases by a microorganism.

In some embodiments, the present invention provides for isolatedmodified polynucleotides encoding modified full-length proteases whereinthe portion of the polynucleotide sequence that encodes the pro regionof the full-length precursor protease comprises mutations encodingsubstitutions at least at one amino acid position chosen from positionsequivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and244. In some embodiments the modified full-length protease is a serineprotease, wherein the portion of the polynucleotide sequence thatencodes the pro region of the full-length precursor protease comprisesmutations encoding substitutions at least at one amino acid positionchosen from positions equivalent to amino acid positions 28-108 and 109of SEQ ID NO:5, 13 and 244. In other embodiments, the full-lengthprotease is an alkaline serine protease derived from a wild-type orvariant precursor alkaline serine protease, wherein the portion of thepolynucleotide sequence that encodes the pro region of the full-lengthprecursor protease comprises mutations encoding substitutions at leastat one amino acid position chosen from positions equivalent to aminoacid positions 28-108 and 109 of SEQ ID NO:5, 13 and 244. In yet otherembodiments, the full-length protease is an alkaline serine proteasederived from a wild-type or variant precursor alkaline serine proteasethat is a B. Clausii or a B. lentus alkaline serine protease, whereinthe portion of the polynucleotide sequence that encodes the pro regionof the full-length precursor protease comprises mutations encodingsubstitutions at least at one amino acid position chosen from positionsequivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and244. In some embodiments, the isolated modified polynucleotide comprisesthe polynucleotide sequence set forth in SEQ ID NOS:1, 9 or 240.

In some embodiments, the present invention provides for isolatedmodified polynucleotides encoding modified full-length proteases whereinthe portion of the polynucleotide sequence that encodes the pro regionof the full-length precursor protease comprises mutations encodingsubstitutions at least at one amino acid position chosen from positionsequivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 and244, wherein the at least one substitution is made at one amino acidposition chosen from E33, E43, A44, E47, V49, E57, A59, E63, E70, E74,E84, and E88 of SEQ ID NO:5, 13 or 244. In some other embodiments, theamino acid substitution of E33 is chosen from E33D, E331, E33S, E33N,E33K, E33H, E33Q and E33R. In yet other embodiments, the amino acidsubstitution of E57 is selected from E57F, E57W, E57K, E57R, E57D, E57M,E57C, E57Q, E57S, E57H and E57N.

In some embodiments, the invention provides isolated modifiedpolynucleotides comprising a precursor pro sequence that has beenmutated to encode substitutions at least at one amino acid positionchosen from positions equivalent to amino acid positions 28-108 and 109of SEQ ID NO:5, 13 or 244. In some additional embodiments, the isolatedmodified polynucleotides further comprise a polynucleotide encoding afull-length protease comprising a mature region, wherein thepolynucleotide encodes a mature region of the protease that is at leastabout 70% identical to SEQ ID NOS:8, 16 or 247.

In some embodiments, the invention provides at least one vectorcomprising modified polynucleotides encoding modified full-lengthproteases wherein the portion of the polynucleotide sequence thatencodes the pro region of the full-length precursor protease comprisesmutations encoding substitutions at least at one amino acid positionchosen from positions equivalent to amino acid positions 28-108 and 109of SEQ ID NO:5, 13 and 244, or isolated modified polynucleotidescomprising a precursor pro sequence that has been mutated to encodesubstitutions at least at one amino acid position chosen from positionsequivalent to amino acid positions 28-108 and 109 of SEQ ID NO:5, 13 or244.

In other embodiments, the invention provides a host cell that istransformed with the vector comprising the modified polynucleotides ofthe invention. In some preferred embodiments, the transformed host cellis a microorganism. For example, in some embodiments, the inventionprovides a host cell that is transformed with at least one vectorcomprising modified polynucleotides encoding modified full-lengthproteases wherein the portion of the polynucleotide sequence thatencodes the pro region of the full-length precursor protease comprisesmutations encoding substitutions at least at one amino acid positionchosen from positions equivalent to amino acid positions 28-108 and 109of SEQ ID NO:5, 13 and 244, or that is transformed with an isolatedmodified polynucleotides comprising a precursor pro sequence that hasbeen mutated to encode substitutions at least at one amino acid positionchosen from positions equivalent to amino acid positions 28-108 and 109of SEQ ID NO:5, 13 or 244. In some embodiments, the host cell that istransformed with the polynucleotides of the invention is a microorganismis chosen from the group consisting of Bacillus sp., Streptomyces sp.,Escherichia sp. and Aspergillus sp. In some other embodiments, the hostcell is B. subtilis.

In some embodiments, the invention provides proteases produced by thetransformed host cells of the invention.

The invention also provides methods for producing a heterologousprotease in a microorganism, wherein the method comprises the steps of:(a) culturing a Bacillus host cell under suitable conditions, whereinthe Bacillus host cell comprises a modified polynucleotide encoding amodified protease; and (b) allowing production of the protease by themicroorganism. In some embodiments, the protease produced by theBacillus host is recovered. Any one of the modified polynucleotidesprovided herein finds use in the methods of the invention.

In some embodiments, invention provides methods for producing aheterologous alkaline serine protease in a microorganism, wherein themethod comprises the steps of: (a) culturing a Bacillus host cell undersuitable conditions, wherein the Bacillus host cell comprises a modifiedpolynucleotide encoding a modified protease; and (b) allowing productionof the protease by the microorganism. The invention also providesmethods for producing a heterologous protease in a microorganism,wherein the method comprises the steps of: (a) culturing a Bacillus hostcell under suitable conditions, wherein the Bacillus host cell comprisesa modified polynucleotide encoding a modified protease comprising amature region that is at least about 70% identical to SEQ ID NOS:8, 16or 247; and (b) allowing production of the protease by themicroorganism. In some embodiments, the protease produced by theBacillus host is recovered.

In some embodiments, the invention provides methods for producing aheterologous protease in a microorganism, wherein the method comprisesthe steps of: (a) culturing a Bacillus host cell under suitableconditions, wherein the Bacillus host cell comprises a modifiedpolynucleotide encoding a modified protease that comprises a pro regioncomprising a mutation encoding a substitution at least at one amino acidposition chosen from positions that are equivalent to amino acidpositions 28-108 and 109 of SEQ ID NO:5, 13 or 244.; and (b) allowingproduction of the protease by the microorganism. In other embodiments,the at least one amino acid substitution is chosen from E33D, E331,E33S, E33N, E33K, E33H, E33Q, E33R, E57F, E57W, E57K, E57R, E57D, E57M,E57C, E57Q, E57S, E57H and E57N.

The invention also provides methods for producing in a microorganism aheterologous protease that is a B. clausii or a B. lentus protease,wherein the method comprises the steps of: (a) culturing a Bacillus hostcell under suitable conditions, wherein the Bacillus host cell comprisesa modified polynucleotide encoding a modified protease; and (b) allowingproduction of the protease by the microorganism.

The invention also provides methods for producing a heterologousprotease in a microorganism, wherein the method comprises the steps of:(a) culturing under suitable conditions a Bacillus host cell chosen fromB. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens, B.brevis, B. stearothermophilus, B. clausii, B. alkalophilus, B.halodurans, B. coagulans, B. circulans, B. pumilus, and B.thuringiensis, wherein the Bacillus host cell comprises a modifiedpolynucleotide encoding a modified protease; and (b) allowing productionof the protease by the microorganism. In some embodiments, the host cellis a B. subtilis host cell.

The invention also provides methods for producing a heterologousprotease in a microorganism, wherein the method comprises the steps of:(a) culturing a Bacillus host cell under suitable conditions, whereinthe Bacillus host cell comprises a modified polynucleotide encoding amodified protease; and (b) allowing production of the protease by themicroorganism, and wherein the heterologous protease exhibits a ratio ofproduction of at least 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the full-length polynucleotide sequence of the wild-typeprotease from B. clausii (SEQ ID NO:1). The portion of the sequence thatencodes the pro region of the protease is shown in bold letters.

FIGS. 1 B, C and D show the polynucleotide sequences (SEQ ID NO:2, 3,and 4) respectively encoding the signal peptide (SEQ ID NO:6), the proregion (SEQ ID NO:7) and the mature form (SEQ ID NO:8) of thefull-length B. clausii protease of SEQ ID NO:5.

FIG. 2A shows the full-length polypeptide sequence of the wild-typeprotease from B. clausii (SEQ ID NO:5). The portion of the sequence thatencodes the pro region of the protease is shown in bold letters.

FIGS. 2 B, C and D show the polypeptide sequences respectively encodingthe signal peptide (SEQ ID NO:6), the pro region (SEQ ID NO:7) and themature form (SEQ ID NO:8) of the full-length B. clausii protease of SEQID NO:5.

FIG. 3A shows the full-length polynucleotide sequence of the variantprotease V049 from B. clausii (SEQ ID NO:9). The portion of the sequencethat encodes the pro region of the protease is shown in bold letters.

FIGS. 3 B, C and D show the polynucleotide sequences (SEQ ID NO:10, 11,and 12) respectively encoding the signal peptide (SEQ ID NO:14), the proregion (SEQ ID NO:15) and the mature form (SEQ ID NO:16) of thefull-length variant B. clausii protease of SEQ ID NO:13.

FIG. 4A shows the full-length polypeptide sequence of the variantprotease V049 from B. clausii (SEQ ID NO:13). The portion of thesequence that encodes the pro region of the protease is shown in boldletters.

FIGS. 4 B, C and D show the polypeptide sequences respectively encodingthe signal peptide (SEQ ID NO:14), the pro region (SEQ ID NO:15) and themature form (SEQ ID NO:16) of the full-length variant B. clausiiprotease of SEQ ID NO:13.

FIG. 5 shows an alignment of the amino acid sequences of the B. clausiiwild-type serine protease of SEQ ID NO:5, the B. clausii variant serineprotease of SEQ ID NO:13 and the B. lentus serine protease of SEQ IDNO:244. Differences in amino acids are underlined.

FIG. 6 provides the map of the pXX-049 plasmid vector comprising thevariant serine protease of SEQ ID NO:9.

FIGS. 7A-C show the polynucleotide sequence of the pXX-V049 plasmidvector (SEQ ID NO:17) used in the present invention.

FIG. 8A shows the full-length polynucleotide sequence of the proteasefrom B. lentus (SEQ ID NO:240). The portion of the sequence that encodesthe pro region of the protease is shown in bold letters.

FIGS. 8B, C and D show the polynucleotide sequences (SEQ ID NO:241, 242,and 243) respectively encoding the signal peptide (SEQ ID NO:245), thepro region (SEQ ID NO:246) and the mature form (SEQ ID NO:247) of the B.lentus precursor protease of SEQ ID NO:244.

FIG. 9A shows the full-length polypeptide sequence of the variantprotease GG36 from B. clausii (SEQ ID NO:244). The portion of thesequence that encodes the pro region of the protease is shown in boldletters.

FIGS. 9 B, C and D show the polypeptide sequences respectively encodingthe signal peptide (SEQ ID NO:245), the pro region (SEQ ID NO:246) andthe mature form (SEQ ID NO:247) of the full-length B. lentus protease ofSEQ ID NO:244.

DESCRIPTION OF THE INVENTION

This invention relates to modified polynucleotides encoding modifiedproteases, and methods for altering the production of proteases inmicroorganisms. In particular, the present invention relates to methodsfor altering the production of proteases in microorganisms, such asBacillus species. The invention discloses modified polynucleotides,vectors, modified polypeptides, and processes for enhancing theproduction of proteases.

The present invention provides modified polynucleotides encodingproteases having a mutated pro region, as well as the modified proteasesencoded by the modified polynucleotides, and methods for producing thesame. The modified protease polynucleotides are suitable for expressingthe modified proteases in microorganisms, and processing the matureforms at levels greater than the corresponding precursor proteases. Theproduced proteases find use in the industrial production of enzymes,suitable for use in various industries, including but not limited to thecleaning, animal feed and textile processing industry. The presentinvention also provides means to produce these enzymes. In somepreferred embodiments, the proteases of the present invention are inpure or relatively pure form.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, protein purification, protein engineering, protein and DNAsequencing, and recombinant DNA fields, which are within the skill ofthe art. Such techniques are known to those of skill in the art and aredescribed in numerous texts and reference works (See e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual”, Second Edition (ColdSpring Harbor), [1989]); and Ausubel et al., “Current Protocols inMolecular Biology” [1987]). All patents, patent applications, articlesand publications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For example,Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Markham,The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991)provide those of skill in the art with a general dictionaries of many ofthe terms used in the invention. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceof the present invention, the preferred methods and materials aredescribed herein. Accordingly, the terms defined immediately below aremore fully described by reference to the Specification as a whole. Also,as used herein, the singular “a”, “an” and “the” includes the pluralreference unless the context clearly indicates otherwise. Numeric rangesare inclusive of the numbers defining the range. Unless otherwiseindicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context theyare used by those of skill in the art.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, a number of terms are defined below.

DEFINITIONS

A “modified protease” is a full-length protease that has an amino acidsequence that is derived from the amino acid sequence of a full-length“precursor protease”. The precursor protease may also be referred to as“unmodified protease”. The modified protease differs from its precursorprotease in the pro region. The precursor protease can be anaturally-occurring i.e. wild-type protease, or it can be a variantprotease. It is the pro region of the wild-type or variant protease thatis modified to generate a modified protease. The amino acid sequence ofthe modified protease is said to be “derived” from the precursorprotease amino acid sequence by the substitution, deletion or insertionof one or more amino acids of the pro region of the precursor amino acidsequence. In preferred embodiments, one or more amino acids of the proregion of the precursor protease are substituted to generate themodified protease. Such modification is of the “precursor DNA sequence”which encodes the amino acid sequence of the precursor protease ratherthan manipulation of the precursor protease per se. The modifiedproteases herein encompass the substitution of any of the nineteennaturally occurring amino acids at any one of the amino acid residues ofthe pro region of the precursor protease. In this context, both“modified” and “precursor” proteases are full-length proteasescomprising a signal peptide, a pro region and a mature region. Thepolynucleotides that encode the modified sequence are referred to as“modified polynucleotides”, and the polynucleotides that encode theprecursor protease are referred to as “precursor polynucleotides”.“Precursor polypeptides” and “precursor polynucleotides” can beinterchangeably referred to as “unmodified precursor polypeptides” or“unmodified precursor polynucleotides”, respectively.

“Naturally-occurring” or “wild-type” refers to a protease or apolynucleotide encoding a protease having the unmodified amino acidsequence identical to that found in nature. Naturally occurring enzymesinclude native enzymes, those enzymes naturally expressed or found inthe particular microorganism. A sequence that is wild-type ornaturally-occurring refers to a sequence from which a variant isderived. The wild-type sequence may encode either a homologous orheterologous protein.

As used herein, “variant” refers to a precursor protein which differsfrom its corresponding wild-type protein by the addition of one or moreamino acids to either or both the C- and N-terminal end, substitution ofone or more amino acids at one or a number of different sites in theamino acid sequence, deletion of one or more amino acids at either orboth ends of the protein or at one or more sites in the amino acidsequence, and/or insertion of one or more amino acids at one or moresites in the amino acid sequence. A variant protein in the context ofthe present invention is exemplified by the B. clauisii protease V049(SEQ ID NO:13), which is a variant of the naturally-occurring proteinMaxacal (SEQ ID NO:5). The precursor protein of the variant can be awild-type or variant protein.

As used herein, “equivalent to,” refers to a residue at the enumeratedposition in a protein or peptide, or a residue that is analogous,homologous, or corresponding to an enumerated residue in a protein orpeptide.

The term “production” with reference to a protease, encompasses the twoprocessing steps of a full-length protease including: 1. the removal ofthe signal peptide, which is known to occur during protein secretion;and 2. the removal of the pro region, which creates the active matureform of the enzyme and which is known to occur during the maturationprocess (Wang et al., Biochemistry 37:3165-3171 (1998); Power et al.,Proc Natl Acad Sci USA 83:3096-3100 (1986)).

The term “processed” with reference to a mature protease refers to thematuration process that a full-length protein e.g. a protease, undergoesto become an active mature enzyme.

The terms “activity ratio” and “ratio of production” are usedinterchangeably to refer to the ratio of the enzymatic activity of amature protease that was processed from modified protease to theenzymatic activity of a mature protease that was processed from anunmodified protease.

The term “full-length protein” herein refers to a primary gene productof a gene and comprising a signal peptide, a pro sequence and a maturesequence.

The term “signal sequence” or “signal peptide” refers to any sequence ofnucleotides and/or amino acids which may participate in the secretion ofthe mature or precursor forms of the protein. This definition of signalsequence is a functional one, meant to include all those amino acidsequences encoded by the N-terminal portion of the protein gene, whichparticipate in the effectuation of the secretion of protein.

The term “pro sequence” or “pro region” is an amino acid sequencebetween the signal sequence and mature protease that is necessary forthe secretion/production of the protease. Cleavage of the pro sequencewill result in a mature active protease. To exemplify, a pro region of aprotease of the present invention at least includes the amino acidsequence identical to residues 28-111 of SEQ ID NO:5, 13 or 244.

The terms “mature form” or “mature region” refer to the final functionalportion of the protein. To exemplify, a mature form of the protease ofthe present invention at least includes the amino acid sequenceidentical to residues 112-380 of SEQ ID NO:5, 13 or 244. In thiscontext, the “mature form” is “processed from” a full-length protease,wherein the processing of the full-length protease encompasses theremoval of the signal peptide and the removal of the pro region.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in the host cell. Similarly, a“heterologous polynucleotide” refers to a polynucleotide that does notnaturally occur in the host cell.

As used herein, “homologous protein” refers to a protein or polypeptidenative or naturally occurring in a cell. Similarly, a “homologouspolynucleotide” refers to a polynucleotide that is native or naturallyoccurring in a cell.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene. Inpreferred embodiments, the promoter is appropriate to the host cell inwhich the target gene is being expressed. The promoter, together withother transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) is necessary to express agiven gene. In general, the transcriptional and translational regulatorysequences include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous.

As used herein, the terms “protease,” and “proteolytic activity” referto a protein or peptide exhibiting the ability to hydrolyze peptides orsubstrates having peptide linkages. Many well known procedures exist formeasuring proteolytic activity (Kalisz, “Microbial Proteinases,” In:Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology,[1988]). For example, proteolytic activity may be ascertained bycomparative assays which analyze the produced protease's ability tohydrolyze a commercial substrate. Exemplary substrates useful in suchanalysis of protease or proteolytic activity, include, but are notlimited to di-methyl casein (Sigma C-9801), bovine collagen (SigmaC-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICNBiomedical 902111). Colorimetric assays utilizing these substrates arewell known in the art (See e.g., WO 99/34011; and U.S. Pat. No.6,376,450, both of which are incorporated herein by reference. The AAPFassay (See e.g., Del Mar et al., Anal. Biochem., 99:316-320 [1979]) alsofinds use in determining the production of mature protease. This assaymeasures the rate at which p-nitroaniline is released as the enzymehydrolyzes the soluble synthetic substrate,succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide(sAAPF-pNA). The rate of production of yellow color from the hydrolysisreaction is measured at 410 nm on a spectrophotometer and isproportional to the active enzyme concentration.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length. Theseterms include, but are not limited to, a single-, double-stranded DNA,genomic DNA, cDNA, or a polymer comprising purine and pyrimidine bases,or other natural, chemically, biochemically modified, non-natural orderivatized nucleotide bases. Non-limiting examples of polynucleotidesinclude genes, gene fragments, chromosomal fragments, ESTs, exons,introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, andprimers.

As used herein, the terms “DNA construct” and “transforming DNA” areused interchangeably to refer to DNA used to introduce sequences into ahost cell or organism. The DNA may be generated in vitro by PCR or anyother suitable technique(s) known to those in the art. In particularlypreferred embodiments, the DNA construct comprises a sequence ofinterest (e.g., a modified sequence). In some embodiments, the sequenceis operably linked to additional elements such as control elements(e.g., promoters, etc.). In some embodiments, the DNA constructcomprises sequences homologous to the host cell chromosome. In otherembodiments, the DNA construct comprises non-homologous sequences. Oncethe DNA construct is assembled in vitro it may be used to mutagenize aregion of the host cell chromosome (i.e., replace an endogenous sequencewith a heterologous sequence).

As used herein, the term “vector” refers to a polynucleotide constructdesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, andplasmids. In some embodiments, the polynucleotide construct comprises aDNA sequence encoding the full-length protease (e.g., modified proteaseor unmodified precursor protease).

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in some eukaryotes orprokaryotes, or integrates into the host chromosome.

As used herein in the context of introducing a nucleic acid sequenceinto a cell, the term “introduced” refers to any method suitable fortransferring the nucleic acid sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, conjugation, and transduction (See e.g.,Ferrari et al., “Genetics,” in Hardwood et al, (eds.), Bacillus, PlenumPublishing Corp., pages 57-72, [1989]).

As used herein, the terms “transformed” and “stably transformed” refersto a cell that has a non-native (heterologous)polynucleotide sequenceintegrated into its genome or as an episomal plasmid that is maintainedfor at least two generations.

The present invention provides isolated modified polynucleotidesencoding amino acid sequences, encoding modified proteases. The modifiedproteases are obtained by mutating the polynucleotide sequence ofprecursor proteases. Specifically, one or more mutations of thepolynucleotide sequence encoding the pro region of the precursorprotease are made to provide the modified polynucleotides of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to modified polynucleotides encoding modifiedproteases, and methods for altering the production of proteases inmicroorganisms. In particular, the present invention relates to methodsfor altering the production of proteases in microorganisms, such asBacillus species. The invention discloses modified polynucleotides,vectors, modified polypeptides, and processes for enhancing theproduction of proteases.

In some embodiments, the invention provides modified polynucleotidesencoding modified proteases that are derived from wild-type or variantprecursor proteases of animal, vegetable or microbial origin.Polynucleotides encoding precursor proteases that are derived frommicroorganisms are preferred, and comprise polynucleotides encodingwild-type precursor proteases and variant precursor proteases, whichhave been derived from the wild-type forms. In some embodiments, themodified polynucleotides of the invention are derived frompolynucleotides encoding precursor proteases of microbial origin. Theinvention also encompasses polynucleotides encoding modified proteasesthat are derived from polynucleotides encoding protein engineeredprecursors. Polynucleotides encoding serine proteases are the preferredprecursor protease polynucleotides, of which the alkaline microbialprotease polynucleotides are particularly preferred. Serine proteasesare enzymes which catalyze the hydrolysis of peptide bonds in whichthere is an essential serine residue at the active site. Serineproteases have molecular weights in the approximately 25,000 to 30,000range (See, Priest, Bacteriol. Rev., 41:711-753 [1977]). In somepreferred embodiments, polynucleotides encoding subtilisin andsubtilisin variants are preferred precursor serine proteasepolynucleotides. In some embodiments, the invention encompassespolynucleotides encoding modified proteases that have been derived frompolynucleotides of microorganisms such as B. licheniformis, B. subtilis,B. amyloliquefaciens, B. clausii, B. lentus and B. halodurans. A widevariety of Bacillus polynucleotides encoding subtilisins have beenidentified and sequenced, for example, subtilisin 168, subtilisin BPN′,subtilisin Carlsberg, subtilisin DY, subtilisin 147 and subtilisin 309(See e.g., EP 414279 B; WO 89/06279; and Stahl et al., J. Bacteriol.,159:811-818 [1984]; each of which is incorporated by reference in theirentirety). In some embodiments of the present invention, mutant (e.g.,variant) protease polynucleotides serve as precursor polynucleotidesencoding proteins from which the modified proteases of the invention arederived. Numerous references provide examples of variant proteases (Seee.g., WO 99/20770; WO 99/20726; WO 99/20769; WO 89/06279; RE 34,606;U.S. Pat. No. 4,914,031; U.S. Pat. No. 4,980,288; U.S. Pat. No.5,208,158; U.S. Pat. No. 5,310,675; U.S. Pat. No. 5,336,611; U.S. Pat.No. 5,399,283; U.S. Pat. No. 5,441,882; U.S. Pat. No. 5,482,849; U.S.Pat. No. 5,631,217; U.S. Pat. No. 5,665,587; U.S. Pat. No. 5,700,676;U.S. Pat. No. 5,741,694; U.S. Pat. No. 5,858,757; U.S. Pat. No.5,880,080; U.S. Pat. No. 6,197,567; and U.S. Pat. No. 6,218,165; each ofwhich is incorporated by reference in its entirety). In otherembodiments, polynucleotides encompassed by the invention includemodified polynucleotides that are derived from polynucleotides encodingcommercially available proteases. For example, commercially availableproteases include, but are not limited to ALCALASE™, SAVINASE™,PRIMASE™, DURALASE™, ESPERASE™, and KANNASE™ (Novo Nordisk A/S),MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, and PURAFECT OXP™,(Genencor International Inc.). In some embodiments, the inventionencompasses modified polynucleotides that are derived frompolynucleotides encoding precursor proteases from B. clausii. In otherembodiments, the invention encompasses modified polynucleotides that arederived from polynucleotides encoding precursor proteases from B.lentus.

In some embodiments, the modified full-length polynculceotides of theinvention comprise sequences that encode the pro region of precursorproteases that have been mutated. The polynucleotide sequence encodingthe pro region of any suitable precursor protease finds use in thegeneration of one or more modified polynucleotides of the invention. Insome preferred embodiments, the portion of a precursor polynucleotidesequence encoding a pro region is mutated to encode one or more aminoacid substitutions at positions that are equivalent to positions 1-109of the protease of SEQ ID NO:5, 13 or 244. In some embodiments, themodified precursor polynucleotides encode for at least one amino acidsubstitutions in the pro region at positions equivalent to E33, E43,A44, E47, V49, E57, A59, E63, E70, E74, E84, and E88 of SEQ ID NO:5, 13or 244. In some embodiments, the polynucleotide sequence encoding theamino acid at position equivalent to E33 is mutated to encode at leastone of substitutions E33D, E331, E33S, E33N, E33K, E33H, E33Q, or E33R.In some other embodiments, the polynucleotide sequence encoding theamino acid at position equivalent to E57 is mutated to encode one ofsubstitutions E57F, E57W, E57K, E57R, E57D, E57M, E57C, E57Q, E57S,E57H, and/or E57N.

In some embodiments, the precursor B. clausii protease polynucleotide(e.g., SEQ ID NO:1) encodes the wild-type protease (MAXACAL™; SEQ IDNO:5).

(SEQ ID NO: 1) ATGAAGAAACCGTTGGGGAAAATTGTCGCAAGCACCGCACTACTCATTTCTGTTGCTTTTAGITCATCGATCGCATCGGCTGCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAAGCTGTCAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAGTCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCGTTGAGTTAAGCCCAGAAGATGTGGACGCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATGGCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCTGGGACGATTGCTGCTTTAAACAATTCGATTGGCGTTCTTGGCGTAGCACCGAACGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGCAGGGAACAATGGCATGCACGTTGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGGAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCAGAAGCGGCAACACGCTAA (SEQ ID NO: 5)MKKPLGKIVASTALLISVAFSSSIASAAEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR

In other embodiments, the precursor B. clausii protease polynucleotide(e.g. SEQ ID NO:9) encodes a variant protease (e.g., SEQ ID NO:13)

(SEQ ID NO: 13) VRSKKLWIVASTALLISVAFSSSIASAAEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGSGSVSSIAQGLEWAGNNVMHVANLSLGLQAPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEA ATR

In other embodiments, the precursor protease polynucleotide is a B.lentus polynucleotide (e.g. SEQ ID NO:240), which encodes a proteaseGG36 of SEQ ID NO:244.

(SEQ ID NO: 240) GTGAGAAGCAAAAAATTGTGGATCGTCGCGTCGACCGCACTACTCATTTCTGTTGCTTTTAGTTCATCGATCGCATCGGCTGCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAAGCTGTCAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAGTCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCGTTGAGTTAAGCCCAGAAGATGTGGACGCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATGGCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTCTAAACAATTCGATTGGCGTTCTTGGCGTAGCGCCGAGCGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGCTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTG TCAATGCAGAAGCTGCAACTCGT(SEQ ID NO:244) MRSKKLWIVASTALLISVAFSSSIASAAEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTMAQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR

In some embodiments, the present invention provides modified full-lengthpolynucleotides derived from the full-length precursor polynucleotide ofSEQ ID NO:9.

(SEQ ID NO: 9) GTGAGAAGCAAAAAATTGTGGATCGTCGCGTCGACCGCACTACTCATTTCTGTTGCTTTTAGITCATCGATCGCATCGGCTGCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAAGCTGTCAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAGTCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCGTTGAGTTAAGCCCAGAAGATGTGGACGCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATGGCGCAATCGGTACCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCTGGGACGATTGCTGCTTTAAACAATTCGATTGGCGTTCTTGGCGTAGCACCGAACGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGTTATGCACGTTGCTAATTTGAGTTTAGGACTGCAGGCACCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGGAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCAGAAGCGGCAACACGTTAA

The polynucleotide sequence of SEQ ID NO:9 comprises a sequence (SEQ IDNO:10) that, when expressed, is contemplated to encode a signal sequencepeptide (SEQ ID NO:14), which spans amino acids 1-27 of SEQ ID NO:13; anN-terminal pro sequence (SEQ ID NO:11) encoding a pro region sequence(SEQ ID NO:15), which spans amino acid residues 28-111 of SEQ ID NO:13;and a mature serine protease sequence (SEQ ID NO:12) encoding amino acidresidues 112-380 of SEQ ID NO:13 (i.e., SEQ ID NO:16). Thepolynucleotide sequence encoding the first 8 amino acids of the signalpeptide of the protease of SEQ ID NO: 13 is the sequence encoding thefirst 8 amino acids of the B. subtilis AprE protease.

(SEQ ID NO: 10) GTGAGAAGCAAAAAATTGTGGATCGTCGCGTCGACCGCACTACTCATTTCTGTTGCTTTTAGTTCATCGATCGCATCGGCT (SEQ ID NO: 15)AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTM (SEQ ID NO: 14)VRSKKLWIVASTALLISVAFSSSIASA (SEQ IS NO: 11)GCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAAGCTGTCAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAGTCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCGTTGAGTTAAGCCCAGAAGATGTGGACGCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATG (SEQ ID NO:12)GCGCAATCGGTACCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCTGGGACGATTGCTGCTTTAAACAATTCGATTGGCGTTCTTGGCGTAGCACCGAACGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGTTATGCACGTTGCTAATTTGAGTTTAGGACTGCAGGCACCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGGAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCAGAAG CGGCAACACGTTAA(SEQ ID NO: 16) AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGSGSVSSIAQGLEWAGNNVMHVANLSLGLQAPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLG STNLYGSGLVNAEAATR

In other embodiments, the present invention provides modifiedfull-length polynucleotides derived from the wild-type full-lengthprecursor polynucleotide of SEQ ID NO:1. The polynucleotide of SEQ IDNO:1 comprises a sequence SEQ ID NO:2 that, when expressed, iscontemplated to encode a signal sequence peptide (SEQ ID NO:6) which isamino acids 1-27 of SEQ ID NO:5; an N-terminal pro sequence (SEQ IDNO:3), encoding amino acid residues 28-111 of SEQ ID NO:5 (i.e. SEQ IDNO:7); and a wild-type mature serine protease sequence (SEQ ID NO:4)encoding amino acid residues 112-380 of SEQ ID NO:5 (i.e., SEQ IDNO:8)).

(SEQ ID NO: 2) ATGAAGAAACCGTTGGGGAAAATTGTCGCAAGCACCGCACTACTCATTTCTGTTGCTTTTAGTTCATCGATCGCATCGGCT (SEQ ID NO: 6)MKKPLGKIVASTALLISVAFSSSIASA (SEQ ID NO: 3)GCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAAGCTGTCAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAGTCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCGTTGAGTTAAGCCCAGAAGATGTGGACGCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATG (SEQ ID NO: 7)AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTM (SEQ ID NO: 4)GCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCTGGGACGATTGCTGCTTTAAACAATTCGATTGGCGTTCTTGGCGTAGCACCGAACGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCAACGTTGCTAATTTGAGTGCTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGGAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAAT GCAGAAGCGGCAACACGCTAA(SEQ ID NO: 8) RVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR

In other embodiments, the present invention provides modifiedfull-length polynucleotides derived from the variant full-lengthprecursor polynucleotide of SEQ ID NO:240. The polynucleotide of SEQ IDNO:240 comprises a sequence SEQ ID NO:241 that, when expressed, iscontemplated to encode a signal sequence peptide (SEQ ID NO:245) whichis amino acids 1-27 of SEQ ID NO:244; an N-terminal pro sequence (SEQ IDNO:242), encoding amino acid residues 28-111 of SEQ ID NO:244 (i.e. SEQID NO:246); and a wild-type mature serine protease sequence (SEQ IDNO:243) encoding amino acid residues 112-380 of SEQ ID NO:244 (i.e., SEQID NO:247)). The polynucleotide sequence encoding the first 8 aminoacids of the signal peptide of the precursor protease of SEQ ID NO: 244is the sequence encoding the first 8 amino acids of the B. subtilis AprEprotease. The pro portion and the mature portion of the precursor GG36are encoded by the GG36 wild-type sequence.

(SEQ ID NO: 241) GTGAGAAGCAAAAAATTGTGGATCGTCGCGTCGACCGCACTACTCATTTCTGTTGCTTTTAGTTCATCGATCGCATCGGCT (SEQ ID NO: 245)MRSKKLWIVASTALLISVAFSSSIASA (SEQ ID NO: 242)GCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAAGCTGTCAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAGTCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCGTTGAGTTAAGCCCAGAAGATGTGGACGCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATG (SEQ ID NO: 246)AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTM (SEQ ID NO: 243)GCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTCTAAACAATTCGATTGGCGTTCTTGGCGTAGCGCCGAGCGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGCTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCAGAAGCTGCAACTC GTTA (SEQ ID NO: 247)AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR

In preferred embodiments, the modified full-length polynculceotides ofthe invention comprise sequences that encode the pro region of precursorproteases that have been mutated. The polynucleotide sequence encodingthe pro region of any suitable precursor protease finds use in thegeneration of one or more modified polynucleotides of the invention. Insome preferred embodiments, the portion of a precursor polynucleotidesequence encoding a pro region is mutated to encode one or more aminoacid substitutions at positions that are equivalent to positions 1-109of the protease of SEQ ID NO:13. In other embodiments, the substitutionsare made at positions that are equivalent to 1-109 of the protease ofSEQ ID NO:5. In yet other embodiments, the substitutions are made atpositions that are equivalent to positions 1-109 of the protease of SEQID NO:244. In some embodiments, the modified precursor polynucleotidesencode for at least one amino acid substitutions at positions equivalentto E33, E43, A44, E47, V49, E57, A59, E63, E70, E74, E84, and E88 of SEQID NO:5, 13 or 244. In some embodiments, the polynucleotide sequenceencoding the amino acid at position equivalent to E33 is mutated toencode at least one of substitutions E33D, E331, E33S, E33N, E33K, E33H,E33Q, or E33R. In some other embodiments, the polynucleotide sequenceencoding the amino acid at position equivalent to E57 is mutated toencode one of substitutions E57F, E57W, E57K, E57R, E57D, E57M, E57C,E57Q, E57S, E57H, and/or E57N.

As discussed above, in some embodiments, the polynucleotide encoding anyprecursor protease is mutated to generate at least one modifiedpolynucleotide encoding at least one modified protease having a mutatedpro region. In some particularly preferred embodiments, full-lengthpolynucleotides encoding serine proteases find use in the generation ofthe modified polynucleotides of the invention. In some alternativeparticularly preferred embodiments, full-length polynucleotides encodingalkaline serine proteases find use. As discussed above, thepolynucleotides encoding the precursor proteases are from microorganismsincluding but not limited to B. licheniformis, B. subtilis, B.amyloliquefaciens, B. clausii, B. lentus and B. halodurans. Theinvention provides for any one of the In some embodiments, the modifiedpolynucleotides of the invention that encode full-length modifiedproteases comprise sequences that encode mature forms of the proteasesthat share at least about 65% amino acid sequence identity, preferablyat least about 70% amino acid sequence identity, more preferably atleast about 75% amino acid sequence identity, still more preferably atleast about 80% amino acid sequence identity, more preferably at leastabout 85% amino acid sequence identity, even more preferably at leastabout 90% amino acid sequence identity, more preferably at least about92% amino acid sequence identity, yet more preferably at least about 95%amino acid sequence identity, more preferably at least about 97% aminoacid sequence identity, still more preferably at least about 98% aminoacid sequence identity, and most preferably at least about 99% aminoacid sequence identity with the amino acid sequence of the mature formof the precursor protease and have comparable or enhanced productionactivity as compared to the precursor polypeptide. In some embodiments,the modified polynucleotides of the invention that encode thefull-length modified proteases comprise sequences that encode matureforms of the proteases that share at least about 65% amino acid sequenceidentity, preferably at least about 70% amino acid sequence identity,more preferably at least about 75% amino acid sequence identity, stillmore preferably at least about 80% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, even morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, yet morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, still morepreferably at least about 98% amino acid sequence identity, and mostpreferably at least about 99% amino acid sequence identity with theamino acid sequence of SEQ ID NO:8, SEQ ID NO:16 or SEQ ID NO:247.

In some embodiments, the modified polynucleotides encode full-lengthamino acid sequences that share up to 65% amino acid sequence identity,preferably up to about 70% amino acid sequence identity, more preferablyup to about 75% amino acid sequence identity, still more preferably upto about 80% amino acid sequence identity, more preferably up to about85% amino acid sequence identity, even more preferably up to about 90%amino acid sequence identity, more preferably up to about 92% amino acidsequence identity, yet more preferably up to about 95% amino acidsequence identity, more preferably up to about 97% amino acid sequenceidentity, still more preferably up to about 98% amino acid sequenceidentity, and most preferably up to about 99% amino acid sequenceidentity with the amino acid sequence of the precursor protease and havecomparable or enhanced production activity, as compared to the precursorpolypeptide.

As will be understood by the skilled artisan, due to the degeneracy ofthe genetic code, a variety of modified polynucleotides encode modifiedproteases. In some other embodiments of the present invention,polynucleotides comprising a nucleotide sequence having at least about70% sequence identity, at least about 75% sequence identity, at leastabout 80% sequence identity, at least about 85% sequence identity, atleast about 90% sequence identity, at least about 92% sequence identity,at least about 95% sequence identity, at least about 97% sequenceidentity, at least about 98% sequence identity and at least about 99%sequence identity to the polynucleotide sequence of SEQ ID NOS:4, 12, or243 are provided.

In some embodiments, the percent identity shared by polynucleotidesequences is determined by direct comparison of the sequence informationbetween the molecules by aligning the sequences and determining theidentity by methods known in the art. In some embodiments, the percentidentity (e.g., amino acid sequence, nucleic acid sequence, and/or genesequence) is determined by a direct comparison of the sequenceinformation between two molecules by aligning the sequences, countingthe exact number of matches between the two aligned sequences, dividingby the length of the shorter sequence, and multiplying the result by100. Readily available computer programs find use in these analyses,including those described above. Programs for determining nucleotidesequence identity are available in the Wisconsin Sequence AnalysisPackage, Version 8 (Genetics Computer Group, Madison, Wis.) for example,the BESTFIT, FASTA and GAP programs, which also rely on the Smith andWaterman algorithm. These programs are readily utilized with the defaultparameters recommended by the manufacturer and described in theWisconsin Sequence Analysis Package referred to above.

An example of an algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul, etal., J. Mol. Biol., 215:403-410 (1990). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. These initial neighborhood word hits actas starting points to find longer HSPs containing them. The word hitsare expanded in both directions along each of the two sequences beingcompared for as far as the cumulative alignment score can be increased.Extension of the word hits is stopped when: the cumulative alignmentscore falls off by the quantity X from a maximum achieved value; thecumulative score goes to zero or below; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See,Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

The BLAST algorithm then performs a statistical analysis of thesimilarity between two sequences (See e.g., Karlin and Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 [1993]). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a serine proteasenucleic acid of this invention if the smallest sum probability in acomparison of the test nucleic acid to a serine protease nucleic acid isless than about 0.1, more preferably less than about 0.01, and mostpreferably less than about 0.001. Where the test nucleic acid encodes aserine protease polypeptide, it is considered similar to a specifiedserine protease nucleic acid if the comparison results in a smallest sumprobability of less than about 0.5, and more preferably less than about0.2.

In some embodiments of the present invention, sequences were analyzed byBLAST and protein translation sequence tools. In some experiments, thepreferred version was BLAST (Basic BLAST version 2.0). The programchosen was “BlastX”, and the database chosen was “nr.” Standard/defaultparameter values were employed.

Several methods are known in the art that are suitable for generatingmodified polynucleotide sequences of the present invention, includingbut not limited to site-saturation mutagenesis, scanning mutagenesis,insertional mutagenesis, deletion mutagenesis, random mutagenesis,site-directed mutagenesis, and directed-evolution, as well as variousother recombinatorial approaches.

In some embodiments, the modified polynucleotide sequences of theinvention comprise modifications of the sequence encoding the pro regionof the protease that are generated with site directed mutagenesis in atleast one codon. In other preferred embodiments, site directedmutagenesis is performed for two or more codons. In some furtherembodiments, modified polynucleotide sequences that encode the proregion of the modified protease have up to about 40%, up to about 45%,up to about 50%, up to about 55%, up to about 60%, up to about 65%, upto about 70%, up to about 75%, up to about 80%, up to about 85%, up toabout 90%, up to about 95%, up to about 98%, or up to about 99% homologywith the polynucleotide that encodes the pro region of the precursorpolypeptide e.g. SEQ ID NOS:3, 11 or 242. In some alternativeembodiments, the modified polynucleotide is generated in vivo, using anyknown mutagenic procedure such as, for example, radiation,nitrosoguanidine and the like. The desired modified polynucleotidesequence is then isolated and used in the methods provided herein.

In some preferred embodiments, site saturation mutagenesis of the proregion of the precursor protease polynucleotides is accomplished byusing a composition comprising mutagenic primers. Mutagenic primers donot precisely match the precursor polynucleotide, and the mismatch ormismatches in the primers are used to introduce the desired mutationinto the template polynucleotide. Non-mutagenic primers, which matchprecisely the precursor polynucleotide are also included in the primercomposition. By adding a mixture of mutagenic primers and non-mutagenicprimers corresponding to at least one of the mutagenic primers, anucleic acid library in which a variety of mutational patterns arepresented is produced. For example, if it is desired that some of themembers of the mutant nucleic acid library retain their precursorsequence at certain positions while other members are mutant at suchsites, the non-mutagenic primers provide the ability to obtain aspecific level of non-mutant members within the nucleic acid library fora given residue. The methods of the invention employ mutagenic andnon-mutagenic oligonucleotides which are generally between about 10 toabout 50 bases in length, more preferably about 15 to about 45 bases inlength. With respect to corresponding mutagenic and non-mutagenicprimers, it is not necessary that the corresponding oligonucleotides beof identical length, but only that there is overlap in the regioncorresponding to the mutation to be added.

In some embodiments, primers are added in a pre-defined ratio. Forexample, if it is desired that the resulting library have a significantlevel of a certain specific mutation and a lesser amount of a differentmutation at the same or different site, by adjusting the amount ofprimer added, the desired biased library is produced. Alternatively, byadding lesser or greater amounts of non-mutagenic primers, the frequencywith which the corresponding mutation(s) are produced in the mutantnucleic acid library is adjusted. Kits comprising compositions ofprimers for site—directed mutagenesis are commercially available andinclude the QuikChange® site-directed mutagenesis kit (Stratagene, SanDiego, Calif.). In some embodiments, precursor polynucleotides arefurther modified to encode modified proteases that comprise two or moreamino acid substitutions in the pro region. Kits for performingmulti-site directed mutagenesis are also commercially available (e.g.,QuikChange® Multisite, Stratagene, San Diego, Calif.).

In some embodiments, the modified polynucleotides of the invention aregenerated using site-saturation methods. In some other embodiments, eachof the modified polynucleotides comprises at least one amino acidsubstitution in the pro region.

The present invention provides modified full-length precursor proteasesthat are encoded by any one of the modified polynucleotides of theinvention. The invention provides for mature forms of proteases thatwhen processed from modified precursor proteases, can be produced atlevels that are greater than those levels attained by processing of theunmodified precursor proteases.

The invention encompasses full-length proteases that have been modifiedby mutating at least one amino acid of the pro region of a precursorprotease. In some embodiments, the precursor protease is mutated at oneor more amino acids of the pro region that are equivalent to amino acids28-109 of the pro region (SEQ ID NO:15) of the full-length precursorprotease of SEQ ID NO:13, the pro region (SEQ ID NO:7) of thefull-length precursor protease of SEQ ID NO:5, or the pro region (SEQ IDNO:246) of the full-length precursor protease of SEQ ID NO:244. In someembodiments, the amino acid substitutions are made at positionsequivalent to E33, E43, A44, E47, V49, E57, A59, E63, E70, E74, E84and/or E88 of the B. clausii V049 precursor protease (SEQ ID NO:13), ofthe B. clausii wild-type protease (SEQ ID NO:5), or of the wild-type B.lentus protease (SEQ ID NO: 244).

In some embodiments, the substitution of amino acids at positionsequivalent to E33, include, but are not limited to E33D, E331, E33S,E33N, E33K, E33H, E33Q or E33R. In other embodiments, the substitutionof E57, includes, but is not limited to E57F, E57W, E57K, E57R, E57D,E57M, E57C, E57Q, E57G, E57S, E57H or E57N. The present inventionencompasses modified proteases wherein any one amino acid substitutionat each of the amino acid positions equivalent to amino acid 28-109 ofSEQ ID NO:5, 13 or 244 is made with any one of the nineteen naturallyoccurring L-amino acids to enhance the production/secretion of themodified protease in comparison to that of the precursor protease (See,Table 2 and Example 4). The amino acids are referred to herein by thecommonly used and understood one letter code (See e.g., Dale, M. W.(1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd.).

In some embodiments, the modified precursor protease polypeptidescomprise mature regions that share at least about 65% amino acidsequence identity, preferably at least about 70% amino acid sequenceidentity, more preferably at least about 75% amino acid sequenceidentity, still more at least about 80% amino acid sequence identity,more preferably at least about 85% amino acid sequence identity, evenmore preferably at least about 90% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, yet morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, still morepreferably at least about 98% amino acid sequence identity, and mostpreferably at least about 99% to the amino acid sequence shown in SEQ IDNOS:8, 16 or 247, and have comparable or enhanced production activity tothe precursor polypeptide.

As indicated above, in some embodiments, the present invention providesvectors comprising the aforementioned polynucleotides. In some preferredembodiments, the vector is an expression vector in which the DNAsequence encoding the protease of the invention is operably linked toadditional segments required for transcription of the DNA. In somepreferred embodiments, the expression vector is derived from plasmid orviral DNA, or in alternative embodiments, contains elements of both.Exemplary vectors include, but are not limited to pXX, pC194, pJH101,pE194, pHP13 (Harwood and Cutting (eds), Molecular Biological Methodsfor Bacillus, John Wiley & Sons, [1990], in particular, chapter 3;suitable replicating plasmids for B. subtilis include those listed onpage 92; Perego, M. (1993) Integrational Vectors for GeneticManipulations in Bacillus subtilis, p. 615-624; A. L. Sonenshein, J. A.Hoch, and R. Losick (ed.), Bacillus subtilis and other Gram-positivebacteria: biochemistry, physiology and molecular genetics, AmericanSociety for Microbiology, Washington, D.C.).

In some preferred embodiments, the vector pXX finds use in theconstruction of vectors comprising the polynucleotides described herein(e.g., pXX-049; See, FIG. 6). It is intended that each of the vectorsdescribed herein will find use in the present invention. In someembodiments, the construct is present on a replicating plasmid (e.g.,pHP13), while in other embodiments, it is integrated into the chromosomein one or more copies. Examples of sites for integration include, butare not limited to the aprE, the amyE, the veg or the pps regions.Indeed, it is contemplated that other sites known to those skilled inthe art will find use in the present invention. In some embodiments, thepromoter is the wild-type promoter for the selected precursor protease.In some other embodiments, the promoter is heterologous to the precursorprotease, but is functional in the host cell. Specifically, examples ofsuitable promoters for use in bacterial host cells include but are notlimited to the pSPAC, pAprE, pAmyE, pVeg, pHpaII promoters, the promoterof the B. stearothermophilus maltogenic amylase gene, the B.amyloliquefaciens (BAN) amylase gene, the B. subtilis alkaline proteasegene, the B. clausii alkaline protease gene the B. pumilus xylosidasegene, the B. thuringiensis crylIIA, and the B. licheniformisalpha-amylase gene. Additional promoters include, but are not limited tothe A4 promoter, as well as phage Lambda P_(R) or P_(L) promoters, andthe E. coli lac, trp or tac promoters.

In some preferred embodiments, the expression vector contains a multiplecloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In some further preferred embodiments, thevector also comprises one or more selectable markers (e.g.,antimicrobial markers such as erythromycin, actinomycin,chloramphenicol, and/or tetracycline). In yet other embodiments, amulticopy replicating plasmid finds use for integration of the plasmidinto the Bacilllus genomic DNA, using methods known in the art.

For expression and production of protein(s) of interest e.g. a protease,in a cell, at least one expression vector comprising at least one copyof a polynucleotide encoding the modified protease, and preferablycomprising multiple copies, is transformed into the cell underconditions suitable for expression of the protein(s). In someparticularly preferred embodiments, the sequences encoding the proteinof interest e.g. proteases (as well as other sequences included in thevector) are integrated into the genome of the host cell, while in otherembodiments, the plasmids remain as autonomous extra-chromosomalelements within the cell. Thus, the present invention provides bothextrachromosomal elements as well as incoming sequences that areintegrated into the host cell genome.

Precursor and modified proteases are produced in host cells of anysuitable Gram-positive microorganism, including bacteria and fungi. Forexample, in some embodiments, the modified protease is produced in hostcells of fungal and/or bacterial origin. In some embodiments, the hostcells are Bacillus sp., Streptomyces sp., Escherichia sp. or Aspergillussp. In some preferred embodiments, the modified proteases are producedby host cells of the genus Bacillus. Examples of Bacillus host cellsthat find use in the production of the modified proteins of the presentinvention include, but are not limited to B. licheniformis, B. lentus,B. subtilis, B. amyloliquefaciens, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B.pumilus, B. thuringiensis, B. clausii, and B. megaterium, as well asother organisms within the genus Bacillus. In some particularlypreferred embodiments, B. subtilis host cells find use. U.S. Pat. Nos.5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus hoststrains that find use in the present invention, although other suitablestrains find use in the present invention.

Industrial strains that find use in the present invention includenon-recombinant (i.e., wild-type) Bacillus strains, as well as variantsof naturally occurring strain and/or recombinant strains. In somepreferred embodiments, the host strain is a recombinant strain, whereina polynucleotide encoding a polypeptide of interest has been introducedinto the host. In some preferred embodiments, the host strain is a B.subtilis host strain and particularly a recombinant Bacillus subtilishost strain. Numerous B. subtilis strains are known, including but notlimited to 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753through PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211strain(See e.g., Hoch et al., Genetics, 73:215-228 [1973]) (See also, U.S.Pat. No. 4,450,235; U.S. Pat. No. 4,302,544; and EP 0134048; each ofwhich is incorporated by reference in its entirety). The use of B.subtilis as an expression host well known in the art (See e.g., See,Palva et al., Gene 19:81-87 [1982]; Fahnestock and Fischer, J.Bacteriol., 165:796-804 [1986]; and Wang et al., Gene 69:39-47 [1988]).

In some embodiments, a preferred Bacillus host is a Bacillus sp. thatincludes a mutation or deletion in at least one of the following genes,degU, degS, degR and degQ. Preferably the mutation is in a degU gene,and more preferably the mutation is degU(Hy)32. (See e.g., Msadek etal., J. Bacteriol., 172:824-834 [1990]; and Olmos et al., Mol. Gen.Genet., 253:562-567 [1997]). A more particularly preferred host strainis a Bacillus subtilis carrying a degU32(Hy) mutation. In some furtherembodiments, the Bacillus host comprises a mutation or deletion inscoC4, (See, e.g., Caldwell et al., J. Bacteriol., 183:7329-7340[2001]); spoIIE (See, Arigoni et al., Mol. Microbiol., 31:1407-1415[1999]); and/or oppA or other genes of the opp operon (See e.g., Peregoet al., Mol. Microbiol., 5:173-185 [1991]). Indeed, it is contemplatedthat any mutation in the opp operon that causes the same phenotype as amutation in the oppA gene will find use in some embodiments of thealtered Bacillus strain of the present invention. In some embodiments,these mutations occur alone, while in other embodiments, combinations ofmutations are present. In some embodiments, an altered Bacillus that canbe used to produce the modified proteases of the invention is a Bacillushost strain that already includes a mutation in one or more of theabove-mentioned genes (in some embodiments, mutations in other genes arealso present). In some alternative embodiments, an altered Bacillusfurther engineered to include mutations of one or more of theabove-mentioned genes finds use.

Host cells are transformed with modified polynucleotides encoding themodified proteases of the present invention using any suitable methodknown in the art. Whether the modified polynucleotide is incorporatedinto a vector or is used without the presence of plasmid DNA, it isintroduced into a microorganism, in some embodiments, preferably an E.coli cell or a competent Bacillus cell. Methods for introducing DNA intoBacillus cells involving plasmid constructs and transformation ofplasmids into E. coli are well known. In some embodiments, the plasmidsare subsequently isolated from E. coli and transformed into Bacillus.However, it is not essential to use intervening microorganisms such asE. coli, and in some embodiments, a DNA construct or vector is directlyintroduced into a Bacillus host.

Those of skill in the art are well aware of suitable methods forintroducing polynucleotide sequences into Bacillus cells (See e.g.,Ferrari et al., “Genetics,” in Harwood et al. (ed.), Bacillus, PlenumPublishing Corp. [1989], pages 57-72; Saunders et al., J. Bacteriol.,157:718-726 [1984]; Hoch et al., J. Bacteriol., 93:1925-1937 [1967];Mann et al., Current Microbiol., 13:131-135 [1986]; and Holubova, FoliaMicrobiol., 30:97 [1985]; Chang et al., Mol. Gen. Genet., 168:11-115[1979]; Vorobjeva et al., FEMS Microbiol. Lett., 7:261-263 [1980]; Smithet al., Appl. Env. Microbiol., 51:634 [1986]; Fisher et al., Arch.Microbiol., 139:213-217 [1981]; and McDonald, J. Gen. Microbiol.,130:203 [1984]). Indeed, such methods as transformation, includingprotoplast transformation and congression, transduction, and protoplastfusion are known and suited for use in the present invention. Methods oftransformation are particularly preferred to introduce a DNA constructprovided by the present invention into a host cell.

In addition to commonly used methods, in some embodiments, host cellsare directly transformed (i.e., an intermediate cell is not used toamplify, or otherwise process, the DNA construct prior to introductioninto the host cell). Introduction of the DNA construct into the hostcell includes those physical and chemical methods known in the art tointroduce DNA into a host cell without insertion into a plasmid orvector. Such methods include, but are not limited to calcium chlorideprecipitation, electroporation, naked DNA, liposomes and the like. Inadditional embodiments, DNA constructs are co-transformed with aplasmid, without being inserted into the plasmid. In furtherembodiments, a selective marker is deleted from the altered Bacillusstrain by methods known in the art (See, Stahl et al., J. Bacteriol.,158:411-418 [1984]; and Palmeros et al., Gene 247:255-264 [2000]).

As indicated above, in some embodiments of the present invention,nucleic acid encoding at least one modified polypeptide of interest isintroduced into a host cell via an expression vector capable ofreplicating within the host cell. Suitable replicating and integratingplasmids for Bacillus known in the art (See e.g., Harwood and Cutting(eds), Molecular Biological Methods for Bacillus, John Wiley & Sons,[1990], in particular, chapter 3; suitable replicating plasmids for B.subtilis include those listed on page 92). Although there are technicalhurdles, those of skill in the art know that there are severalstrategies for the direct cloning of DNA in Bacillus.

Methods known in the art to transform Bacillus, include such methods asplasmid marker rescue transformation, which involves the uptake of adonor plasmid by competent cells carrying a partially homologousresident plasmid (Contente et al., Plasmid 2:555-571 [1979]; Haima etal., Mol. Gen. Genet., 223:185-191 [1990]; Weinrauch et al., J.Bacteriol., 154:1077-1087 [1983]; and Weinrauch et al., J. Bacteriol.,169:1205-1211 [1987]). In this method, the incoming donor plasmidrecombines with the homologous region of the resident “helper” plasmidin a process that mimics chromosomal transformation.

Other methods involving transformation by protoplast transformation areknown in the art (See e.g., Chang and Cohen, Mol. Gen. Genet.,168:111-115 [1979]; Vorobjeva et al., FEMS Microbiol. Lett., 7:261-263[1980]; Smith et al., Appl. Env. Microbiol., 51:634 [1986]; Fisher etal., Arch. Microbiol., 139:213-217 [1981]; McDonald [1984] J. Gen.Microbiol., 130:203 [1984]; and Bakhiet et al., 49:577 [1985]). Inaddition, Mann et al., (Mann et al., Curr. Microbiol., 13:131-135[1986]) describe transformation of Bacillus protoplasts, and Holubova(Holubova, Microbiol., 30:97 [1985]) describe methods for introducingDNA into protoplasts using DNA-containing liposomes. In some preferredembodiments, marker genes are used in order to indicate whether or notthe gene of interest is present in the host cell.

In addition to these methods, in other embodiments, host cells aredirectly transformed. In “direct transformation,” an intermediate cellis not used to amplify, or otherwise process, the modifiedpolynucleotide prior to introduction into the host (i.e., Bacillus)cell. Introduction of the modified polynucleotide into the host cellincludes those physical and chemical methods known in the art tointroduce modified polynucleotide into a host cell without insertioninto a plasmid or vector. Such methods include but are not limited tothe use of competent cells, as well as the use of “artificial means”such as calcium chloride precipitation, electroporation, etc. tointroduce DNA into cells. Thus, the present invention finds use withnaked DNA, liposomes and the like. In yet other embodiments, themodified polynucleotides are co-transformed with a plasmid without beinginserted into the plasmid.

More particularly, the present invention provides constructs, vectorscomprising polynucleotides described herein, host cells transformed withsuch vectors, proteases expressed by such host cells, expression methodsand systems for the production of homologous or heterologous serineprotease enzymes derived from microorganisms (in particular, members ofthe genus Bacillus). In some embodiments, the modified polynucleotide(s)encoding modified serine protease(s) are used to produce recombinanthost cells suitable for the expression of the modified serineprotease(s). In some preferred embodiments, the expression hosts arecapable of enhancing the secretion of the mature forms of the modifiedprotease(s) thus increasing the commercial production of proteases.

In some embodiments, the host cells and transformed cells of the presentinvention are cultured in conventional nutrient media. The suitablespecific culture conditions, such as temperature, pH and the like areknown to those skilled in the art. In addition, some preferred cultureconditions may be found in the scientific literature such as Hopwood(2000) Practical Streptomyces Genetics, John Innes Foundation, NorwichUK; Hardwood et al., (1990) Molecular Biological Methods for Bacillus,John Wiley and from the American Type Culture Collection (ATCC).

In some embodiments, host cells transformed with polynucleotidesequences encoding modified proteases are cultured under conditionssuitable for the expression and recovery of the encoded protein fromcell culture. The protein produced by a recombinant host cell comprisinga modified protease of the present invention is secreted into theculture media. In some embodiments, other recombinant constructions jointhe heterologous or homologous polynucleotide sequences to nucleotidesequence encoding a protease polypeptide domain which facilitatespurification of the soluble proteins (Kroll D J et al (1993) DNA CellBiol 12:441-53).

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath J (1992) Protein Expr Purif3:263-281), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego Calif.) between the purification domain and the heterologousprotein also find use to facilitate purification.

In some preferred embodiments, the cells transformed with polynucleotidesequences encoding heterologous or homologous protein or endogenouslyhaving said protein are cultured under conditions suitable for theexpression and recovery of the encoded protein from the cell culturemedium. In some embodiments, other recombinant constructions includeheterologous or homologous polynucleotide sequences to nucleotidesequence encoding a polypeptide domain which facilitates purification ofsoluble protein (e.g., tags of various sorts) (Kroll et al., DNA Cell.Biol., 12:441-53 [1993]).

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath, Prot. Expr. Purif.,3:263-281 [1992]), protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle Wash.).The inclusion of a cleavable linker sequence such as Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and the heterologous protein finds use in facilitatingpurification.

In some preferred embodiments, the transformed host cells of the presentinvention are cultured in a suitable nutrient medium under conditionspermitting the expression of the present protease, after which theresulting protease is recovered from the culture. The medium used toculture the cells comprises any conventional medium suitable for growingthe host cells, such as minimal or complex media containing appropriatesupplements. Suitable media are available from commercial suppliers ormay be prepared according to published recipes (e.g., in catalogues ofthe American Type Culture Collection). In some embodiments, the proteaseproduced by the cells is recovered from the culture medium byconventional procedures, including, but not limited to separating thehost cells from the medium by centrifugation or filtration,precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt (e.g., ammonium sulfate), chromatographicpurification (e.g., ion exchange, gel filtration, affinity, etc.). Thus,any method suitable for recovering the protease(s) of the presentinvention finds use in the present invention. Indeed, it is not intendedthat the present invention be limited to any particular purificationmethod.

As indicated above, the polypeptides of the invention are produced asmature enzymes at levels greater than the mature enzymes processes fromtheir corresponding unmodified precursor polypeptides. In preferredembodiments of the present invention, the mutations within the proregion of a precursor polypeptide enhance the secretion/expression ofthe mature polypeptide when compared to a corresponding mature proteasethat has been processed from a precursor protease when produced by aBacillus strain under the same conditions.

One measure of enhancement can be determined as an activity ratio, whichcan be expressed as the ratio of the enzymatic activity of the matureform processed from the modified protease to the enzymatic activity ofthe mature form processed from the precursor protease. A ratio equal orgreater than 1 indicates that the mature form of modified protease isproduced at levels equal or greater than those at which the mature formof precursor protease is produced. For example, an activity ratio of 1.5indicates that the mature protease that has been processed from amodified protease is produced at 1.5 times the level at which the matureprotease that is processed from the precursor protease i.e. the modifiedprotease yields 50% more mature protease than the unmodified precursorprotease. In some embodiments, the activity ratio is at least 1, atleast about 1.05, about at least about 1.1, at least about 1.2, at leastabout 1.3, at least about 1.4, at least about 1.5, at least about 1.6,at least about 1.7, at least about 1.8. at least about 1.9, and at leastabout 2. In other embodiments, the activity ratio is at least about 2.1,at least about 2.2, at least about 2.3, at least about 2.4, at leastabout 2.5, at least about 2.6, at least about 2.7, at least about 2.8,at least about 2.9 and at least about 3. In yet other embodiments, theactivity ratio is at least about 3.5, at least about 4.0, and at leastabout 5. Thus, in some embodiments, production of the mature proteaseprocessed from the modified protease is enhanced by at least about 0.5%,about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4.0%,about 5.0%, about 8.0%, about 10%, about 15%, about 20%, about 25%,about 30%, about 40%, about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 100% or more,compared to the corresponding mature protease processed from theunmodified precursor protease. In other embodiments, production of themature form of the protease that is processed from the modified proteaseis enhanced by at least about 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190%, and up to at least about 200%, or more compared to thecorresponding production of the mature form of the protease that wasprocessed from the unmodified precursor protease. In some embodiments,the enhanced production of the modified protease is determined based onthe ratio of the proteolytic activity of the mature form processed fromthe modified protease compared to the proteolytic activity of the matureform of the corresponding unmodified precursor protease.

Other means for determining the levels of secretion of a heterologous orhomologous protein in a host cell and detecting secreted proteinsinclude, but are not limited to methods that use either polyclonal ormonoclonal antibodies specific for the protein. Examples include, butare not limited to enzyme-linked immunosorbent assays (ELISA),radioimmunoassays (RIA), fluorescent immunoassays (FIA), and fluorescentactivated cell sorting (FACS). These and other assays are well known inthe art (See e.g., Maddox et al., J. Exp. Med., 158:1211 [1983]). Insome preferred embodiments of the present invention, secretion is higherusing the methods and compositions provided herein than when using thesame methods or compositions, but where a peptide transport protein orgene product of a peptide transport operon has not been introduced.

There are various assays known to those of ordinary skill in the art fordetecting and measuring activity of polypeptides of the invention. Inparticular, assays are available for measuring protease activity thatare based on the release of acid-soluble peptides from casein orhemoglobin, measured as absorbance at 280 nm or colorimetrically usingthe Folin method (See e.g., Bergmeyer et al., “Methods of EnzymaticAnalysis” vol. 5, Peptidases, Proteinases and their Inhibitors, VerlagChemie, Weinheim [1984]). Some other assays involve the solubilizationof chromogenic substrates (See e.g., Ward, “Proteinases,” in Fogarty(ed.)., Microbial Enzymes and Biotechnology, Applied Science, London,[1983], pp 251-317). Other exemplary assays include, but are not limitedto succinyl-Ala-Ala-Pro-Phe-para nitroanilide assay (SAAPFpNA) and the2,4,6-trinitrobenzene sulfonate sodium salt assay (TNBS assay). Numerousadditional references known to those in the art provide suitable methods(See e.g., Wells et al., Nucleic Acids Res. 11:7911-7925 [1983];Christianson et al., Anal. Biochem., 223:119-129 [1994]; and Hsia etal., Anal Biochem., 242:221-227 [1999]). It is not intended that thepresent invention be limited to any particular assay method(s).

In some embodiments, the production of the modified protease by amicroorganism is determined by using a ratio of the activity of a matureprotease processed from a modified precursor protease compared to theactivity of the mature protease processed from an unmodified precursorprotease. In some particularly preferred embodiments, ratio of 1 orgreater is desired.

Other means for determining the levels of production of a protein ofinterest e.g. a protease, in a host cell and detecting expressedproteins include the use of immunoassays with either polyclonal ormonoclonal antibodies specific for the protein. Examples includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),fluorescence immunoassay (FIA), and fluorescent activated cell sorting(FACS). However, other methods are known to those in the art and finduse in assessing the protein of interest (See e.g., Hampton et al.,Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn.[1990]; and Maddox et al., J. Exp. Med., 158:1211 [1983]). In somepreferred embodiments, secretion of a protein of interest is higher inthe altered strain obtained using the present invention than in acorresponding unaltered host. As known in the art, the altered Bacilluscells produced using the present invention are maintained and grownunder conditions suitable for the expression and recovery of apolypeptide of interest from cell culture (See e.g., Hardwood andCutting (eds.) Molecular Biological Methods for Bacillus, John Wiley &Sons [1990]). It is not intended that the present invention be limitedto any particular assay method(s).

All publications and patents mentioned herein are herein incorporated byreference. Various modifications and variations of the described methodand system of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as should not beunduly limited to such specific embodiments. Indeed, variousmodifications of the described modes for carrying out the invention thatare obvious to those skilled in the art and/or related fields areintended to be within the scope of the present invention.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: ppm (parts per million); M (molar); mM(millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg(milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL(milliliters); μl and μL (microliters); cm (centimeters); mm(millimeters); μm (micrometers); nm (nanometers); U (units); V (volts);MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) andhr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient);ND (not done); NA (not applicable); rpm (revolutions per minute); H₂O(water); dH₂O (deionized water); (HCl (hydrochloric acid); aa (aminoacid); by (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copyor complementary DNA); DNA (deoxyribonucleic acid); ssDNA (singlestranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotidetriphosphate); RNA (ribonucleic acid); MgCl₂ (magnesium chloride); NaCl(sodium chloride); w/v (weight to volume); v/v (volume to volume); g(gravity); OD (optical density); Dulbecco's phosphate buffered solution(DPBS); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (optical density at 600nm); A₄₀₅ (absorbance at 405 nm); PAGE (polyacrylamide gelelectrophoresis); PBS (phosphate buffered saline [150 mM NaCl, 10 mMsodium phosphate buffer, pH 7.2]); PBST (PBS+0.25% TWEEN®-20); PEG(polyethylene glycol); PCR (polymerase chain reaction); SDS (sodiumdodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); SDS (sodium dodecylsulfate); bME, BME and βME(beta-mercaptoethanol or 2-mercaptoethanol); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine(N-Rris-(hydroxymethyl)-methyl]-glycine); DMSO (dimethyl sulfoxide); Taq(Thermus aquaticus DNA polymerase); Klenow (DNA polymerase I large(Klenow) fragment); rpm (revolutions per minute); EGTA (ethyleneglycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid); EDTA(ethylenediaminetetracetic acid); bla (β-lactamase orampicillin-resistance gene); DNA2.0 (DNA2.0, Menlo Park, Calif.); OXOID(Oxoid, Basingstoke, Hampshire, UK); Corning (Corning Life Sciences,Corning, N.Y.); ATCC (American Type Culture Collection, Rockville, Md.);Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma Chemical Co.,St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Pisacataway, N.J.); NCBI(National Center for Biotechnology Information); Applied Biosystems(Applied Biosystems, Foster City, Calif.); Clontech (CLONTECHLaboratories, Palo Alto, Calif.); Operon Technologies (OperonTechnologies, Inc., Alameda, Calif.); Bachem (Bachem Bioscience, Inc.,King of Prussia, Pa.); Difco (Difco Laboratories, Detroit, Mich.); GIBCOBRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Millipore(Millipore, Billerica, Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.);Invitrogen (Invitrogen Corp., San Diego, Calif.); NEB (New EnglandBiolabs, Beverly, Mass.); Sigma (Sigma Chemical Co., St. Louis, Mo.);Pierce (Pierce Biotechnology, Rockford, Ill.); Takara (Takara Bio Inc.Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EM Science(EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.);Molecular Devices (Molecular Devices, Corp., Sunnyvale, Calif.); R&DSystems (R&D Systems, Minneapolis, Minn.); Stratagene (StratageneCloning Systems, La Jolla, Calif.); and Microsoft (Microsoft, Inc.,Redmond, Wash.).

Example 1 Site-Scanning Mutagenesis of the SigP and Pro Sequence of theBacillus clausii Alkaline Protease V049

Site-saturation mutagenesis of the pro sequence of the Bacillus clausiialkaline protease V049 precursor protease was performed using theQuikChange® site-directed mutagenesis kit (QC; Stratagene) according tothe directions provided by the manufacturer. Site-saturation librarieswere created to include modified polynucleotides sequences of theprecursor polynucleotide encoding the B. clausii protease variant V049.The sequence encoding the pro region of the V049 polynucleotidecontained in the pXX-V049 plasmid (FIGS. 5 and 6; SEQ ID NO:17) wasmutated to produce a library of polynucleotides each comprising amutation of one codon of the pro region of the precursor protease. Eachcodon of the pro region of V049 exemplified by NNG/C was mutated to besubstituted by the 32 possible nucleotide triplets that encode the 20naturally occurring amino acids. Complementary overlapping primers weredesigned for each codon of interest with 18 bases flanking the NNScodon, and the sequences of the primers (SEQ ID NO: 18-239) are given inTable 1.

TABLE 1 Bases Bases SEQ Primer* Primer Sequence left** right*** ID NOV049-1F TTAAAAGGAGAGGGTAAAGANNSAGAAGCAAAAAATTGTG 20 17  18 V049-2FGGAGAGGGTAAAGAGTGNNSAGCAAAAAATTGTGGATC 17 18  19 V049-3FGAGGGTAAAGAGTGAGANNSAAAAAATTGTGGATCGTC 17 18  20 V049-4FGGTAAAGAGTGAGAAGCNNSAAATTGTGGATCGTCGC 17 17  21 V049-5FGTAAAGAGTGAGAAGCAAANNSTTGTGGATCGTCGCGTC 19 17  22 V049-6FGAGTGAGAAGCAAAAAANNSTGGATCGTCGCGTCGAC 17 17  23 V049-7FGTGAGAAGCAAAAAATTGNNSATCGTCGCGTCGACCGC 18 17  24 V049-8FGAAGCAAAAAATTGTGGNNSGTCGCGTCGACCGCACTAC 17 19  25 V049-9FGCAAAAAATTGTGGATCNNSGCGTCGACCGCACTACTC 17 18  26 V049-10FCAAAAAATTGTGGATCGTCNNSTCGACCGCACTACTCATTTC 19 20  27 V049-11FAAAAATTGTGGATCGTCGCGNNSACCGCACTACTCATTTC 20 17  28 V049-12FAATTGTGGATCGTCGCGTCGNNSGCACTACTCATTTCTGTTG 20 19  29 V049-13FGGATCGTCGCGTCGACCNNSCTACTCATTTCTGTTGC 17 17  30 V049-14FGATCGTCGCGTCGACCGCANNSCTCATTTCTGTTGCTTTTAG 19 20  31 V049-15FGTCGCGTCGACCGCACTANNSATTTCTGTTGCTTTTAG 18 17  32 V049-16FCGTCGACCGCACTACTCNNSTCTGTTGCTTTTAGTTC 17 17  33 V049-17FCGACCGCACTACTCATTNNSGTTGCTTTTAGTTCATC 17 17  34 V049-18FCCGCACTACTCATTTCTNNSGCTTTTAGTTCATCGATC 17 18  35 V049-19FCACTACTCATTTCTGTTNNSTTTAGTTCATCGATCGC 17 17  36 V049-20FCTACTCATTTCTGTTGCTNNSAGTTCATCGATCGCATC 18 17  37 V049-21FCTCATTTCTGTTGCTTTTNNSTCATCGATCGCATCGGC 18 17  38 V049-22FCATTTCTGTTGCTTTTAGTNNSTCGATCGCATCGGCTGC 19 17  39 V049-23FCTGTTGCTTTTAGTTCANNSATCGCATCGGCTGCTGAAG 17 19  40 V049-24FGTTGCTTTTAGTTCATCGNNSGCATCGGCTGCTGAAGAAG 18 19  41 V049-25FCTTTTAGTTCATCGATCNNSTCGGCTGCTGAAGAAGC 17 17  42 V049-26FCTTTTAGTTCATCGATCGCANNSGCTGCTGAAGAAGCAAAAG 20 19  43 V049-27FGTTCATCGATCGCATCGNNSGCTGAAGAAGCAAAAGAAAA 17 20  44 V049-28FCATCGATCGCATCGGCTNNSGAAGAAGCAAAAGAAAAATA 17 20  45 V049-29FCGATCGCATCGGCTGCTNNSGAAGCAAAAGAAAAATATTT 17 20  46 V049-30FGATCGCATCGGCTGCTGAANNSGCAAAAGAAAAATATTTAAT 19 20  47 V049-31FCATCGGCTGCTGAAGAANNSAAAGAAAAATATTTAATTG 17 19  48 V049-32FCGGCTGCTGAAGAAGCANNSGAAAAATATTTAATTGG 17 17  49 V049-33FCTGCTGAAGAAGCAAAANNSAAATATTTAATTGGCTTTAA 17 20  50 V049-34FCTGAAGAAGCAAAAGAANNSTATTTAATTGGCTTTAATG 17 19  51 V049-35FGAAGAAGCAAAAGAAAAANNSTTAATTGGCTTTAATGAG 18 18  52 V049-36FGAAGCAAAAGAAAAATATNNSATTGGCTTTAATGAGCAG 18 18  53 V049-37FCAAAAGAAAAATATTTANNSGGCTTTAATGAGCAGGAAG 17 19  54 V049-38FCAAAAGAAAAATATTTAATTNNSTTTAATGAGCAGGAAGC 20 17  55 V049-39FGAAAAATATTTAATTGGCNNSAATGAGCAGGAAGCTGTC 18 18  56 V049-40FAAAAATATTTAATTGGCTTTNNSGAGCAGGAAGCTGTCAG 20 17  57 V049-41FAATATTTAATTGGCTTTAATNNSCAGGAAGCTGTCAGTGAG 20 18  58 V049-42FATTTAATTGGCTTTAATGAGNNSGAAGCTGTCAGTGAGTTTG 20 19  59 V049-43FTAATTGGCTTTAATGAGCAGNNSGCTGTCAGTGAGTTTGTAG 20 19  60 V049-44FGCTTTAATGAGCAGGAANNSGTCAGTGAGTTTGTAGAAC 17 19  61 V049-45FCTTTAATGAGCAGGAAGCTNNSAGTGAGTTTGTAGAACAAG 19 19  62 V049-46FTTAATGAGCAGGAAGCTGTCNNSGAGTTTGTAGAACAAGTAG 20 19  63 V049-47FGAGCAGGAAGCTGTCAGTNNSTTTGTAGAACAAGTAGAG 18 18  64 V049-48FCAGGAAGCTGTCAGTGAGNNSGTAGAACAAGTAGAGGC 18 17  65 V049-49FGAAGCTGTCAGTGAGTTTNNSGAACAAGTAGAGGCAAATG 18 19  66 V049-50FCTGTCAGTGAGTTTGTANNSCAAGTAGAGGCAAATGAC 17 18  67 V049-51FGTCAGTGAGTTTGTAGAANNSGTAGAGGCAAATGACGAG 18 18  68 V049-52FGTGAGTTTGTAGAACAANNSGAGGCAAATGACGAGGTC 17 18  69 V049-53FGAGTTTGTAGAACAAGTANNSGCAAATGACGAGGTCGC 18 17  70 V049-54FGTTTGTAGAACAAGTAGAGNNSAATGACGAGGTCGCCATTC 19 19  71 V049-55FGTAGAACAAGTAGAGGCANNSGACGAGGTCGCCATTCTC 18 18  72 V049-56FGAACAAGTAGAGGCAAATNNSGAGGTCGCCATTCTCTC 18 17  73 V049-57FCAAGTAGAGGCAAATGACNNSGTCGCCATTCTCTCTGAG 18 18  74 V049-58FGTAGAGGCAAATGACGAGNNSGCAATTCTCTCTGAGGAAG 18 19  75 V049-59FGAGGCAAATGACGAGGTCNNSATTCTCTCTGAGGAAGAG 18 18  76 V049-60FCAAATGACGAGGTCGCCNNSCTCTCTGAGGAAGAGGAAG 17 19  77 V049-61FCAAATGACGAGGTCGCCATTNNSTCTGAGGAAGAGGAAGTC 20 18  78 V049-62FGACGAGGTCGCCATTCTCNNSGAGGAAGAGGAAGTCGAAAT 18 20  79 V049-63FGAGGTCGCCATTCTCTCTNNSGAAGAGGAAGTCGAAATTG 18 19  80 V049-64FGTCGCCATTCTCTCTGAGNNSGAGGAAGTCGAAATTGAATT 18 20  81 V049-65FCCATTCTCTCTGAGGAANNSGAAGTCGAAATTGAATTG 17 18  82 V049-66FCATTCTCTCTGAGGAAGAGNNSGTCGAAATTGAATTGCTTC 19 19  83 V049-67FCTCTCTGAGGAAGAGGAANNSGAAATTGAATTGCTTCATG 18 19  84 V049-68FCTGAGGAAGAGGAAGTCNNSATTGAATTGCTTCATGAATT 17 20  85 V049-69FGAGGAAGAGGAAGTCGAANNSGAATTGCTTCATGAATTTG 18 19  86 V049-70FGAAGAGGAAGTCGAAATTNNSTTGCTTCATGAATTTGAAAC 18 20  87 V049-71FGAGGAAGTCGAAATTGAANNSCTTCATGAATTTGAAAC 18 17  88 V049-72FGAAGTCGAAATTGAATTGNNSCATGAATTTGAAACGATTC 18 19  89 V049-73FGTCGAAATTGAATTGCTTNNSGAATTTGAAACGATTCC 18 17  90 V049-74FGAAATTGAATTGCTTCATNNSTTTGAAACGATTCCTGTTTT 18 20  91 V049-75FAAATTGAATTGCTTCATGAANNSGAAACGATTCCTGTTTTATC 20 20  92 V049-76FGAATTGCTTCATGAATTTNNSACGATTCCTGTTTTATC 18 17  93 V049-77FAATTGCTTCATGAATTTGAANNSATTCCTGTTTTATCCGTTG 20 19  94 V049-78FCTTCATGAATTTGAAACGNNSCCTGTTTTATCCGTTGAG 18 18  95 V049-79FCATGAATTTGAAACGATTNNSGTTTTATCCGTTGAGTTAAG 18 20  96 V049-80FGAATTTGAAACGATTCCTNNSTTATCCGTTGAGTTAAG 18 17  97 V049-81FAATTTGAAACGATTCCTGTTNNSTCCGTTGAGTTAAGCCC 20 17  98 V049-82FGAAACGATTCCTGTTTTANNSGTTGAGTTAAGCCCAGAAG 18 19  99 V049-83FCGATTCCTGTTTTATCCNNSGAGTTAAGCCCAGAAGATG 17 19 100 V049-84FGATTCCTGTTTTATCCGTTNNSTTAAGCCCAGAAGATGTG 19 18 101 V049-85FCTGTTTTATCCGTTGAGNNSAGCCCAGAAGATGTGGAC 17 18 102 V049-86FGTTTTATCCGTTGAGTTANNSCCAGAAGATGTGGACGC 18 17 103 V049-87FTTTTATCCGTTGAGTTAAGCNNSGAAGATGTGGACGCGCTTG 20 19 104 V049-88FCCGTTGAGTTAAGCCCANNSGATGTGGACGCGCTTGAAC 17 19 105 V049-89FGTTGAGTTAAGCCCAGAANNSGTGGACGCGCTTGAACTC 18 18 106 V049-90FGAGTTAAGCCCAGAAGATNNSGACGCGCTTGAACTCGATC 18 19 107 V049-91FGTTAAGCCCAGAAGATGTGNNSGCGCTTGAACTCGATCC 19 17 108 V049-92FGCCCAGAAGATGTGGACNNSCTTGAACTCGATCCAGC 17 17 109 V049-93FCAGAAGATGTGGACGCGNNSGAACTCGATCCAGCGATTTC 17 20 110 V049-94FGAAGATGTGGACGCGCTTNNSCTCGATCCAGCGATTTC 18 17 111 V049-95FGATGTGGACGCGCTTGAANNSGATCCAGCGATTTCTTATAT 18 20 112 V049-96FGTGGACGCGCTTGAACTCNNSCCAGCGATTTCTTATATTG 18 19 113 V049-97FGACGCGCTTGAACTCGATNNSGCGATTTCTTATATTGAAG 18 19 114 V049-98FCGCTTGAACTCGATCCANNSATTTCTTATATTGAAGAG 17 18 115 V049-99FCTTGAACTCGATCCAGCGNNSTCTTATATTGAAGAGGATG 18 19 116 V049-100FGAACTCGATCCAGCGATTNNSTATATTGAAGAGGATGC 18 17 117 V049-101FCTCGATCCAGCGATTTCTNNSATTGAAGAGGATGCAGAAG 18 19 118 V049-102FGATCCAGCGATTTCTTATNNSGAAGAGGATGCAGAAGTAAC 18 20 119 V049-103FCAGCGATTTCTTATATTNNSGAGGATGCAGAAGTAAC 17 17 120 V049-104FCGATTTCTTATATTGAANNSGATGCAGAAGTAACGAC 17 17 121 V049-105FGATTTCTTATATTGAAGAGNNSGCAGAAGTAACGACAATG 19 18 122 V049-106FCTTATATTGAAGAGGATNNSGAAGTAACGACAATGGC 17 17 123 V049-107FCTTATATTGAAGAGGATGCANNSGTAACGACAATGGCGCAATC 20 20 124 V049-108FATATTGAAGAGGATGCAGAANNSACGACAATGGCGCAATC 20 17 125 V049-109FGAAGAGGATGCAGAAGTANNSACAATGGCGCAATCGGTAC 18 19 126 V049-110FGAGGATGCAGAAGTAACGNNSATGGCGCAATCGGTACC 18 17 127 V049-111FGATGCAGAAGTAACGACANNSGCGCAATCGGTACCATG 18 17 128 V049-1RCACAATTTTTTGCTTCTSNNTCTTTACCCTCTCCTTTTAA 17 20 129 V049-2RGATCCACAATTTTTTGCTSNNCACTCTTTACCCTCTCC 18 17 130 V049-3RGACGATCCACAATTTTTTSNNTCTCACTCTTTACCCTC 18 17 131 V049-4RGCGACGATCCACAATTTSNNGCTTCTCACTCTTTACC 17 17 132 V049-5RGACGCGACGATCCACAASNNTTTGCTTCTCACTCTTTAC 17 19 133 V049-6RGTCGACGCGACGATCCASNNTTTTTTGCTTCTCACTC 17 17 134 V049-7RGCGGTCGACGCGACGATSNNCAATTTTTTGCTTCTCAC 17 18 135 V049-8RGTAGTGCGGTCGACGCGACSNNCCACAATTTTTTGCTTC 19 17 136 V049-9RGAGTAGTGCGGTCGACGCSNNGATCCACAATTTTTTGC 18 17 137 V049-10RGAAATGAGTAGTGCGGTCGASNNGACGATCCACAATTTTTTG 20 19 138 V049-11RGAAATGAGTAGTGCGGTSNNCGCGACGATCCACAATTTTT 17 20 139 V049-12RCAACAGAAATGAGTAGTGCSNNCGACGCGACGATCCACAATT 19 20 140 V049-13RGCAACAGAAATGAGTAGSNNGGTCGACGCGACGATCC 17 17 141 V049-14RCTAAAAGCAACAGAAATGAGSNNTGCGGTCGACGCGACGATC 20 19 142 V049-15RCTAAAAGCAACAGAAATSNNTAGTGCGGTCGACGCGAC 17 18 143 V049-16RGAACTAAAAGCAACAGASNNGAGTAGTGCGGTCGACG 17 17 144 V049-17RGATGAACTAAAAGCAACSNNAATGAGTAGTGCGGTCG 17 17 145 V049-18RGATCGATGAACTAAAAGCSNNAGAAATGAGTAGTGCGG 18 17 146 V049-19RGCGATCGATGAACTAAASNNAACAGAAATGAGTAGTG 17 17 147 V049-20RGATGCGATCGATGAACTSNNAGCAACAGAAATGAGTAG 17 18 148 V049-21RGCCGATGCGATCGATGASNNAAAAGCAACAGAAATGAG 17 18 149 V049-22RGCAGCCGATGCGATCGASNNACTAAAAGCAACAGAAATG 17 19 150 V049-23RCTTCAGCAGCCGATGCGATSNNTGAACTAAAAGCAACAG 19 17 151 V049-24RCTTCTTCAGCAGCCGATGCSNNCGATGAACTAAAAGCAAC 19 18 152 V049-25RGCTTCTTCAGCAGCCGASNNGATCGATGAACTAAAAG 17 17 153 V049-26RCTTTTGCTTCTTCAGCAGCSNNTGCGATCGATGAACTAAAAG 19 20 154 V049-27RTTTTCTTTTGCTTCTTCAGCSNNCGATGCGATCGATGAAC 20 17 155 V049-28RTATTTTTCTTTTGCTTCTTCSNNAGCCGATGCGATCGATG 20 17 156 V049-29RAAATATTTTTCTTTTGCTTCSNNAGCAGCCGATGCGATCG 20 17 157 V049-30RATTAAATATTTTTCTTTTGCSNNTTCAGCAGCCGATGCGATC 20 19 158 V049-31RCAATTAAATATTTTTCTTTSNNTTCTTCAGCAGCCGATG 19 17 159 V049-32RCCAATTAAATATTTTTCSNNTGCTTCTTCAGCAGCCG 17 17 160 V049-33RTTAAAGCCAATTAAATATTTSNNTTTTGCTTCTTCAGCAG 20 17 161 V049-34RCATTAAAGCCAATTAAATASNNTTCTTTTGCTTCTTCAG 19 17 162 V049-35RCTCATTAAAGCCAATTAASNNTTTTTCTTTTGCTTCTTC 18 18 163 V049-36RCTGCTCATTAAAGCCAATSNNATATTTTTCTTTTGCTTC 18 18 164 V049-37RCTTCCTGCTCATTAAAGCCSNNTAAATATTTTTCTTTTG 19 17 165 V049-38RGCTTCCTGCTCATTAAASNNAATTAAATATTTTTCTTTTG 17 20 166 V049-39RGACAGCTTCCTGCTCATTSNNGCCAATTAAATATTTTTC 18 18 167 V049-40RCTGACAGCTTCCTGCTCSNNAAAGCCAATTAAATATTTTT 17 20 168 V049-41RCTCACTGACAGCTTCCTGSNNATTAAAGCCAATTAAATATT 18 20 169 V049-42RCAAACTCACTGACAGCTTCSNNCTCATTAAAGCCAATTAAAT 19 20 170 V049-43RCTACAAACTCACTGACAGCSNNCTGCTCATTAAAGCCAATTA 19 20 171 V049-44RGTTCTACAAACTCACTGACSNNTTCCTGCTCATTAAAGC 19 17 172 V049-45RCTTGTTCTACAAACTCACTSNNAGCTTCCTGCTCATTAAAG 19 19 173 V049-46RCTACTTGTTCTACAAACTCSNNGACAGCTTCCTGCTCATTAA 19 20 174 V049-47RCTCTACTTGTTCTACAAASNNACTGACAGCTTCCTGCTC 18 18 175 V049-48RGCCTCTACTTGTTCTACSNNCTCACTGACAGCTTCCTG 17 18 176 V049-49RCATTTGCCTCTACTTGTTCSNNAAACTCACTGACAGCTTC 19 18 177 V049-50RGTCATTTGCCTCTACTTGSNNTACAAACTCACTGACAG 18 17 178 V049-51RCTCGTCATTTGCCTCTACSNNTTCTACAAACTCACTGAC 18 18 179 V049-52RGACCTCGTCATTTGCCTCSNNTTGTTCTACAAACTCAC 18 17 180 V049-53RGCGACCTCGTCATTTGCSNNTACTTGTTCTACAAACTC 17 18 181 V049-54RGAATGGCGACCTCGTCATTSNNCTCTACTTGTTCTACAAAC 19 19 182 V049-55RGAGAATGGCGACCTCGTCSNNTGCCTCTACTTGTTCTAC 18 18 183 V049-56RGAGAGAATGGCGACCTCSNNATTTGCCTCTACTTGTTC 17 18 184 V049-57RCTCAGAGAGAATGGCGACSNNGTCATTTGCCTCTACTTG 18 18 185 V049-58RCTTCCTCAGAGAGAATGGCSNNCTCGTCATTTGCCTCTAC 19 18 186 V049-59RCTCTTCCTCAGAGAGAATSNNGACCTCGTCATTTGCCTC 18 18 187 V049-60RCTTCCTCTTCCTCAGAGAGSNNGGCGACCTCGTCATTTG 19 17 188 V049-61RGACTTCCTCTTCCTCAGASNNAATGGCGACCTCGTCATTTG 18 20 189 V049-62RATTTCGACTTCCTCTTCCTCSNNGAGAATGGCGACCTCGTC 20 18 190 V049-63RCAATTTCGACTTCCTCTTCSNNAGAGAGAATGGCGACCTC 19 18 191 V049-64RAATTCAATTTCGACTTCCTCSNNCTCAGAGAGAATGGCGAC 20 18 192 V049-65RCAATTCAATTTCGACTTCSNNTTCCTCAGAGAGAATGG 18 17 193 V049-66RGAAGCAATTCAATTTCGACSNNCTCTTCCTCAGAGAGAATG 19 19 194 V049-67RCATGAAGCAATTCAATTTCSNNTTCCTCTTCCTCAGAGAG 19 18 195 V049-68RAATTCATGAAGCAATTCAATSNNGACTTCCTCTTCCTCAG 20 17 196 V049-69RCAAATTCATGAAGCAATTCSNNTTCGACTTCCTCTTCCTC 19 18 197 V049-70RGTTTCAAATTCATGAAGCAASNNAATTTCGACTTCCTCTTC 20 18 198 V049-71RGTTTCAAATTCATGAAGSNNTTCAATTTCGACTTCCTC 17 18 199 V049-72RGAATCGTTTCAAATTCATGSNNCAATTCAATTTCGACTTC 19 18 200 V049-73RGGAATCGTTTCAAATTCSNNAAGCAATTCAATTTCGAC 17 18 201 V049-74RAAAACAGGAATCGTTTCAAASNNATGAAGCAATTCAATTTC 20 18 202 V049-75RGATAAAACAGGAATCGTTTCSNNTTCATGAAGCAATTCAATTT 20 20 203 V049-76RGATAAAACAGGAATCGTSNNAAATTCATGAAGCAATTC 17 18 204 V049-77RCAACGGATAAAACAGGAATSNNTTCAAATTCATGAAGCAATT 19 20 205 V049-78RCTCAACGGATAAAACAGGSNNCGTTTCAAATTCATGAAG 18 18 206 V049-79RCTTAACTCAACGGATAAAACSNNAATCGTTTCAAATTCATG 20 18 207 V049-80RCTTAACTCAACGGATAASNNAGGAATCGTTTCAAATTC 17 18 208 V049-81RGGGCTTAACTCAACGGASNNAACAGGAATCGTTTCAAATT 17 20 209 V049-82RCTTCTGGGCTTAACTCAACSNNTAAAACAGGAATCGTTTC 19 18 210 V049-83RCATCTTCTGGGCTTAACTCSNNGGATAAAACAGGAATCG 19 17 211 V049-84RCACATCTTCTGGGCTTAASNNAACGGATAAAACAGGAATC 18 19 212 V049-85RGTCCACATCTTCTGGGCTSNNCTCAACGGATAAAACAG 18 17 213 V049-86RGCGTCCACATCTTCTGGSNNTAACTCAACGGATAAAAC 17 18 214 V049-87RCAAGCGCGTCCACATCTTCSNNGCTTAACTCAACGGATAAAA 19 20 215 V049-88RGTTCAAGCGCGTCCACATCSNNTGGGCTTAACTCAACGG 19 17 216 V049-89RGAGTTCAAGCGCGTCCACSNNTTCTGGGCTTAACTCAAC 18 18 217 V049-90RGATCGAGTTCAAGCGCGTCSNNATCTTCTGGGCTTAACTC 19 18 218 V049-91RGGATCGAGTTCAAGCGCSNNCACATCTTCTGGGCTTAAC 17 19 219 V049-92RGCTGGATCGAGTTCAAGSNNGTCCACATCTTCTGGGC 17 17 220 V049-93RGAAATCGCTGGATCGAGTTCSNNCGCGTCCACATCTTCTG 20 17 221 V049-94RGAAATCGCTGGATCGAGSNNAAGCGCGTCCACATCTTC 17 18 222 V049-95RATATAAGAAATCGCTGGATCSNNTTCAAGCGCGTCCACATC 20 18 223 V049-96RCAATATAAGAAATCGCTGGSNNGAGTTCAAGCGCGTCCAC 19 18 224 V049-97RCTTCAATATAAGAAATCGCSNNATCGAGTTCAAGCGCGTC 19 18 225 V049-98RCTCTTCAATATAAGAAATSNNTGGATCGAGTTCAAGCG 18 17 226 V049-99RCATCCTCTTCAATATAAGASNNCGCTGGATCGAGTTCAAG 19 18 227 V049-100RGCATCCTCTTCAATATASNNAATCGCTGGATCGAGTTC 17 18 228 V049-101RCTTCTGCATCCTCTTCAATSNNAGAAATCGCTGGATCGAG 19 18 229 V049-102RGTTACTTCTGCATCCTCTTCSNNATAAGAAATCGCTGGATC 20 18 230 V049-103RGTTACTTCTGCATCCTCSNNAATATAAGAAATCGCTG 17 17 231 V049-104RGTCGTTACTTCTGCATCSNNTTCAATATAAGAAATCG 17 17 232 V049-105RCATTGTCGTTACTTCTGCSNNCTCTTCAATATAAGAAATC 18 19 233 V049-106RGCCATTGTCGTTACTTCSNNATCCTCTTCAATATAAG 17 17 234 V049-107RGATTGCGCCATTGTCGTTACSNNTGCATCCTCTTCAATATAAG 20 20 235 V049-108RGATTGCGCCATTGTCGTSNNTTCTGCATCCTCTTCAATAT 17 20 236 V049-109RGTACCGATTGCGCCATTGTSNNTACTTCTGCATCCTCTTC 19 18 237 V049-110RGGTACCGATTGCGCCATSNNCGTTACTTCTGCATCCTC 17 18 238 V049-111RCATGGTACCGATTGCGCSNNTGTCGTTACTTCTGCATC 17 18 239

*The primer names provided reflect the amino acid position of thesubstitution; “R” indicates that the primer is the reverse primer and“F” indicates that the primer is a forward primer. For example,V049-108F is the forward primer that was used in the substitution ofamino acid at position 108 of the V049 precursor protease.

**“Bases left” and ***“Bases Right” indicate the number of bases to theleft and to the right of the mutating codon that are present in theprimer. These bases are complementary to the bases of the templateprecursor polynucleotide bases (i.e. V049).

Polynucleotide sequence of vector pXX-049 SEQ ID NO: 17AATTCCTCCATTTTCTTCTGCTATCAAAATAACAGACTCGTGATTTTCCAAACGAGCTTTCAAAAAAGCCTCTGCCCCTTGCAAATCGGATGCCTGTCTATAAAATTCCCGATATTGGCTTAAACAGCGGCGCAATGGCGGCCGCATCTGATGTCTTTGCTTGGCGAATGTTCATCTTATTTCTTCCTCCCTCTCAATAATTTTTTCATTCTATCCCTTTTCTGTAAAGTTTATTTTTCAGAATACTTTTATCATCATGCTTTGAAAAAATATCACGATAATATCCATTGTTCTCACGGAAGCACACGCAGGTCATTTGAACGAATTTTTTCGACAGGAATTTGCCGGGACTCAGGAGCATTTAACCTAAAAAAGCATGACATTTCAGCATAATGAACATTTACTCATGTCTATTTTCGTTCTTTTCTGTATGAAAATAGTTATTTCGAGTCTCTACGGAAATAGCGAGAGATGATATACCTAAATAGAGATAAAATCATCTCAAAAAAATGGGTCTACTAAAATATTATTCCATCTATTACAATAAATTCACAGAATAGTCTTTTAAGTAAGTCTACTCTGAATTTTTTTAAAAGGAGAGGGTAAAGAGTGAGAAGCAAAAAATTGTGGATCGTCGCGTCGACCGCACTACTCATTTCTGTTGCTTTTAGTTCATCGATCGCATCGGCTGCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAAGCTGTCAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAGTCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCGTTGAGTTAAGCCCAGAAGATGTGGACGCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATGGCGCAATCGGTACCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCTGGGACGATTGCTGCTTTAAACAATTCGATTGGCGTTCTTGGCGTAGCACCGAACGCGGAACTATACGCTGTTAAAGTATTAGGGGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGTTATGCACGTTGCTAATTTGAGTTTAGGACTGCAGGCACCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGGAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAGCGGACTTGTCAATGCAGAAGCGGCAACACGTTAATCAATAAAAAAACGCTGTGCGGTTAAAGGGCACAGCGTTTTTTTGTGTATGAATCGGGATCCTCGATCGAGACTAGAGTCGATTTTTACAAGAATTAGCTTTATATAATTTCTGTTTTTCTAAAGTTTTATCAGCTACAAAAGACAGAAATGTATTGCAATCTTCAACTAAATCCATTTGATTCTCTCCAATATGACGTTTAATAAATTTCTGAAATACTTGATTTCTTTGTTTTTTCTCAGTATACTTTTCCATGTTATAACACATAAAAACAACTTAGTTTTCACAAACTATGACAATAAAAAAAGTTGCTTTTTCCCCTTTCTATGTATGTTTTTTACTAGTCATTTAAAACGATACATTAATAGGTACGAAAAAGCAACTTTTTTTGCGCTTAAAACCAGTCATACCAATAACTTAAGGGTAACTAGCCTCGCCGGCAATAGTTACCCTTATTATCAAGATAAGAAAGAAAAGGATTTTTCGCTACGCTCAAATCCTTTAAAAAAACACAAAAGACCACATTTTTTAATGTGGTCTTTATTCTTCAACTAAAGCACCCATTAGTTCAACAAACGAAAATTGGATAAAGTGGGATATTTTTAAAATATATATTTATGTTACAGTAATATTGACTTTTAAAAAAGGATTGATTCTAATGAAGAAAGCAGACAAGTAAGCCTCCTAAATTCACTTTAGATAAAAATTTAGGAGGCATATCAAATGAACTTTAATAAAATTGATTTAGACAATTGGAAGAGAAAAGAGATATTTAATCATTATTTGAACCAACAAACGACTTTTAGTATAACCACAGAAATTGATATTAGTGTTTTATACCGAAACATAAAACAAGAAGGATATAAATTTTACCCTGCATTTATTTTCTTAGTGACAAGGGTGATAAACTCAAATACAGCTTTTAGAACTGGTTACAATAGCGACGGAGAGTTAGGTTATTGGGATAAGTTAGAGCCACTTTATACAATTTTTGATGGTGTATCTAAAACATTCTCTGGTATTTGGACTCCTGTAAAGAATGACTTCAAAGAGTTTTATGATTTATACCTTTCTGATGTAGAGAAATATAATGGTTCGGGGAAATTGTTTCCCAAAACACCTATACCTGAAAATGCTTTTTCTCTTTCTATTATTCCATGGACTTCATTTACTGGGTTTAACTTAAATATCAATAATAATAGTAATTACCTtCTACCCATTATTACAGCAGGAAAATTCATTAATAAAGGTAATTCAATATATTTACCGCTATCTTTACAGGTACATCATTCTGTTTGTGATGGTTATCATGCAGGATTGTTTATGAACTCTATTCAGGAATTGTCAGATAGGCCTAATGACTGGCTTTTATAATATGAGATAATGCCGACTGTACTTTTTACAGTCGGTTTTCTAATGTCACTAACCTGCCCCGTTAGTTGAAGAAGGTTTTTATATTACAGCTCCAGATCCATATCCTTCTTTTTCTGAACCGACTTCTCCTTTTTCGCTTCTTTATTCCAATTGCTTTATTGACGTTGAGCCTCGGAACCCTTAACAATCCCAAAACTTGTCGAATGGTCGGCTTAATAGCTCACGCTATGCCGACATTCGTCTGCAAGTTTAGTTAAGGGTTCTTCTCAACGCACAATAAATTTTCTCGGCATAAATGCGTGGTCTAATTTTTATTTTTAATAACCTTGATAGCAAAAAATGCCATTCCAATACAAAACCACATACCTATAATCGACCTGCAGGAATTAATTCCTCCATTTTCTTCTGCTATCAAAATAACAGACTCGTGATTTTCCAAACGAGCTTTCAAAAAAGCCTCTGCCCCTTGCAAATCGGATGCCTGTCTATAAAATTCCCGATATTGGCTTAAACAGCGGCGCAATGGCGGCCGCATCTGATGTCTTTGCTTGGCGAATGTTCATCTTATTTCTTCCTCCCTCTCAATAATTTTTTCATTCTATCCCTTTTCTGTAAAGTTTATTTTTCAGAATACTTTTATCATCATGCTTTGAAAAAATATCACGATAATATCCATTGTTCTCACGGAAGCACACGCAGGTCATTTGAACGAATTTTTTCGACAGGAATTTGCCGGGACTCAGGAGCATTTAACCTAAAAAAGCATGACATTTCAGCATAATGAACATTTACTCATGTCTATTTTCGTTCTTTTCTGTATGAAAATAGTTATTTCGAGTCTCTACGGAAATAGCGAGAGATGATATACCTAAATAGAGATAAAATCATCTCAAAAAAATGGGTCTACTAAAATATTATTCCATCTATTACAATAAATTCACAGAATAGTCTTTTAAGTAAGTCTACTCTGAATTTTTTTATCAAGCTAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGGGTATTGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCTGCCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAACCTAACTACGGCTACACTAGAAGGTGGTGGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAACTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTAGTGCCACATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCTTCAAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGC CAGTG

The QC reaction consisted of 40.25 μL of sterile distilled H₂O, 5 μL ofPfuTurbo 10× buffer from the kit, 1 μL dNTPs from the kit, 1.25 μL offorward primer (100 ng/μL), 1.25 μL reverse primer (100 ng/μL), 0.25 μLof pMSAT-NcoI miniprep DNA as template (˜50 ng), and 1 μL of PfuTurbofrom the kit, for a total of 50 μL. The cycling conditions were 95° C.for 1 min, once, followed by 19-20 cycles of 95° C. for 1 min., 55° C.for 1 min, and 68° C. for 12 min. To analyze the reaction, 5 μL of thereaction was run on a 1.2% E-gel (Invitrogen) upon completion. Next,DpnI digestion was carried out twice sequentially, with 1 μL and 1 μL ofenzyme at 37° C. for 2 to 8 hours. A negative control was carried outunder similar conditions, but without any primers. Then, 1 μL of theDpnI-digested reaction product was transformed into 50 μL of one-shotTOP10 electrocompetent cells (Invitrogen) using a BioRad electroporator.Then, 1 ml of SOC provided with the TOP10 cells (Invitrogen) were addedto the electroporated cells and incubated with shaking for 1 hour beforeplating on LA plates containing 5 ppm chloramphenicol. The plates wereincubated at 37° C. overnight. After this incubation, 96 colonies fromeach of the libraries (i.e., each site) were inoculated in 200 μL of LBcontaining 10-50 ppm of Chloramphenicol in 96-well microtiter plates andgrow overnight at 37° C. The plates were frozen at −80° C. afteraddition of glycerol to 20% final concentration the next day, and theywere used for high throughput sequencing with the V049SEQ-R2 primers.

Similarly, mutations of codons encoding two or more amino acids of thepro region of the precursor protease V049 are performed using theQuikChange® Multi Site-Directed mutagenesis (QCMS; Stratagene). The QCMSreaction is performed using 19.25 μL of sterile distilled H2O, 2.5 μL of10× buffer from the kit, 1 μL dNTPs from the kit, 1 μL of 5′phosphorylated forward primer (100 ng/μL), 0.25 μL of pMSAT-NcoIminiprep DNA as template (˜50 ng), and 1 μL of the enzyme blend from thekit for a total of 25 μL. The cycling conditions are 95° C. for 1 minonce, followed by 30 cycles of 95° C. for 1 min, 55° C. for 1 min, and65° C. for 12 min. To analyze the reaction product, 2.5 μL of thereaction are run on a 1.2% E-gel (Invitrogen) upon completion. Next,DpnI digestion is carried out twice sequentially, with 1 ul and then 0.5μL of enzyme at 37° C. for 2 to 8 hours. The controls, transformation,and sequencing are performed as for the QC method described above.

Example 2 Host Cell Transformation and Expression of Modified Proteases

Plasmids pXX-049 containing polynucleotides encoding the modifiedproteins of interest were digested twice with DpnI for 3-5 hours at 37C.

Transformation and Screening in E. coli

1 ul of DpnI digested plasmid DNA was used to transform E. coli Top10cells by electroporation. The transformed cells were plated onto LB agarplates containing 5 ppm CMP (chloramphenicol), and colonies were allowedto grow overnight. 96 individual colonies were picked and transferred tocorresponding wells of a 96-well micro-titer plate containing LB+5 ppmCMP+50 ppm carbenicillin. Cultures were grown overnight at 37° C. whileshaking at 250 rpm. Glycerol stock was added in the culture to a finalconcentration of 10%. Plasmid DNA was prepared from the 96 E. colicultures, and a portion of the plasmid DNA preparation was sequenced(Cogenics, Morrisville, N.C.). Automated sequence analysis was performedusing Phrep, Phrap, Consed, Custal W software.

Transformation into Bacillus substilis

A second portion of the plasmid DNA was used to transform B. subtilishost cells. Two microliters of plasmid DNA from each of the 96 E. colicultures carrying the appropriate mutations were used to transform 100ul of B. substilis ComK competent cells. The cells were incubated at 37°C. for 45 minutes while shaking at 250 rpm. Cells from each of the 96transformation mixtures were plated onto LA containing 1.6% skim milkand 5 ppm CMP and incubated overnight in a 37° C. incubator.

Four colonies, from each of the 96 transformations, were picked andindividually transferred to a micro-titer plate containing 150 ul of LBand 5 ppm CMP per well. A number of wells of the micro-titer platecontained the appropriate controls. The micro-titer plates were thenincubated for four hours at 37° C. while rotating at 250 rpm. 10 ul ofeach of the cultures were transferred to a new micro-titer platecontaining 140 ul of Grant II media+5 ppm CMP, pH 7.3. GrantII media wasprepared as follows: Solution I: 10 g of Soytone were dissolved in 500ml water and autoclaved for 20-25 minutes; Solution II: 3 ml of 1MK2HPO4, 75 g glucose, 3.6 g urea, 100 ml Grant's 10× MOPS were dilutedinto 400 ml water. Solutions I and II were mixed and the pH adjusted topH7.3 with HCl/NaOH. The final volume was adjusted to 1 L, and the finalsolution was sterilized through 0.22-um PES filter.

The micro-titer plate cultures were incubated in shaker at 37° C., 250rpm. Samples were taken at regular intervals (up to 40 hours) for assayanalysis.

Example 3 Measurement of Modified Protease Production: AAPF Assay ofProtease Activity

Each of the B. subtilis cultures obtained as described in Example 2, wasassayed for the production of the modified proteases. The enzymesproduced were assayed for activity against the substrate, succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanalide (AAPF). The assay measured theproduction of modified protease as the increase in absorbance at 405nm/min resulting from the hydrolysis and release of p-nitroanaline(Estell et al., J Biol. Chem., 260:6518-6521 (1985)). The measurementswere made using the Sofmax Pro software, and the specified conditionswere set as: Type: Kinetic; Reduction: Vmax Points (Read best 15/28points); Lm1: 405 nm; Time: 5 minutes; and Interval: 11 Seconds. Tenmicroliters of each of the B. subtilis cultures were diluted 100 ul ofTris Buffer, containing 10 mM Tris+0.005% TWEEN®-80, pH 8.6; and 25 ulof 100 mg/ml AAPF. The relative activity of each of the modifiedproteases was calculated, and the effect of each amino acid substitutionon the production of the corresponding modified protease was determinedas a ratio of the activity of the mature protease processed from eachmodified protease to the activity of the mature protease processed fromthe unmodified V049 precursor protease. The results are given in Table2.

Once the DNA construct was stably integrated into a competent Bacillussubtilis strain, the activity of the modified proteases was measured inmicrotiter assays and the activity was compared to the activity of thecorresponding precursor protease.

Ten microliters of overnight Grant II Media cell cultures were dilutedto 100 ul of Tris Buffer, containing 10 mM Tris+0.005% TWEEN®-80 pH 8.6;and 25 ul of 100 mg/ml AAPF substrate were used to assay for proteaseactivity. Assays were done in microtiter plates and the Softmax ProSoftware was used.

The results showed that amino acid substitution of most of the aminoacids of the precursor V049 protease lead to an enhanced production ofthe mature form of the protease. In addition, site saturation of each ofthe substituted amino acids showed that each amino acid can besubstituted by two or more amino acids at the same position to increasethe production of the mature form relative to that obtained from theprecursor protease having unmodified pro region.

TABLE 2 VARIANT PROTEIN VARIANT AMINO CON- ACTIVITY POSITION* CODE ACIDCENTRATION RATIO# 1 V001D D 102.37 1.02 1 V001Q Q 98.52 0.99 1 V001F F96.85 0.97 1 V001L L 58.70 0.59 1 V001A A 58.67 0.59 1 V001H H 36.540.37 1 V001I I 35.67 0.36 1 V001G G 29.93 0.30 1 V001Y Y 14.97 0.15 1V001E E 9.30 0.09 1 V001T T 6.99 0.07 1 V001C C 6.75 0.07 1 V001W W 6.610.07 1 V001S S 5.54 0.06 1 V001R R 5.19 0.05 2 R002M M 122.01 1.22 2R002W W 104.55 1.05 2 R002K K 103.46 1.03 2 R002C C 101.20 1.01 2 R002SS 101.19 1.01 2 R002L L 98.36 0.98 2 R002F F 93.71 0.94 2 R002H H 93.650.94 2 R002N N 89.23 0.89 2 R002A A 82.16 0.82 2 R002G G 73.95 0.74 2R002D D 60.97 0.61 2 R002V V 53.99 0.54 2 R002T T 40.33 0.40 2 R002Y Y13.66 0.14 3 S003F F 133.20 1.33 3 S003M M 127.94 1.28 3 S003R R 121.001.21 3 S003T T 116.45 1.16 3 S003Q Q 114.41 1.14 3 S003N N 105.57 1.06 3S003I I 104.04 1.04 3 S003V V 101.09 1.01 3 S003W W 99.63 1.00 3 S003G G99.50 0.99 3 S003D D 98.94 0.99 3 S003H H 98.52 0.99 3 S003A A 97.600.98 3 S003P P 93.74 0.94 3 S003Y Y 65.38 0.65 3 S003L L 48.58 0.49 3S003C C 10.43 0.10 4 K004T T 121.17 1.21 4 K004V V 116.61 1.17 4 K004Y Y111.33 1.11 4 K004I I 109.36 1.09 4 K004C C 109.34 1.09 4 K004R R 107.321.07 4 K004F F 103.89 1.04 4 K004H H 96.72 0.97 4 K004A A 95.03 0.95 4K004Q Q 92.30 0.92 4 K004P P 91.49 0.91 4 K004N N 86.73 0.87 4 K004S S78.31 0.78 4 K004G G 53.68 0.54 4 K004E E 29.18 0.29 4 K004L L 7.86 0.085 K005W W 123.75 1.24 5 K005N N 118.59 1.19 5 K005Q Q 114.87 1.15 5K005Y Y 112.41 1.12 5 K005V V 112.29 1.12 5 K005H H 111.16 1.11 5 K005GG 110.84 1.11 5 K005S S 109.37 1.09 5 K005D D 108.39 1.08 5 K005C C103.96 1.04 5 K005R R 100.76 1.01 5 K005T T 99.54 1.00 5 K005A A 91.660.92 5 K005L L 90.96 0.91 5 K005P P 49.40 0.49 5 K005F F 6.26 0.06 5K005M M 5.65 0.06 6 L006M M 122.55 1.23 6 L006S S 118.01 1.18 6 L006G G116.94 1.17 6 L006N N 115.98 1.16 6 L006V V 115.39 1.15 6 L006P P 115.111.15 6 L006H H 114.99 1.15 6 L006D D 114.30 1.14 6 L006K K 111.33 1.11 6L006E E 109.38 1.09 6 L006A A 109.11 1.09 6 L006T T 105.11 1.05 6 L006II 105.10 1.05 6 L006R R 103.58 1.04 6 L006F F 101.84 1.02 6 L006C C101.69 1.02 6 L006W W 77.03 0.77 6 L006Y Y 7.99 0.08 7 W007V V 125.701.26 7 W007M M 121.99 1.22 7 W007S S 121.49 1.21 7 W007R R 121.42 1.21 7W007P P 113.12 1.13 7 W007T T 112.42 1.12 7 W007N N 111.62 1.12 7 W007QQ 110.40 1.10 7 W007F F 109.54 1.10 7 W007K K 99.28 0.99 7 W007C C 99.280.99 7 W007G G 98.93 0.99 7 W007H H 95.65 0.96 7 W007A A 92.24 0.92 7W007L L 80.60 0.81 7 W007Y Y 61.72 0.62 7 W007I I 6.21 0.06 8 I008F F114.74 1.15 8 I008S S 111.06 1.11 8 I008P P 107.32 1.07 8 I008L L 106.571.07 8 I008V V 105.39 1.05 8 I008T T 105.22 1.05 8 I008Y Y 103.53 1.04 8I008M M 100.20 1.00 8 I008E E 99.53 1.00 8 I008A A 95.64 0.96 8 I008D D75.18 0.75 8 I008R R 4.60 0.05 9 V009R R 130.14 1.30 9 V009P P 122.651.23 9 V009M M 114.91 1.15 9 V009I I 114.34 1.14 9 V009Y Y 111.23 1.11 9V009S S 107.52 1.08 9 V009C C 105.80 1.06 9 V009T T 105.04 1.05 9 V009EE 100.29 1.00 9 V009W W 98.90 0.99 9 V009L L 97.67 0.98 9 V009A A 93.840.94 9 V009H H 86.35 0.86 9 V009N N 85.33 0.85 9 V009K K 6.32 0.06 10A010M M 127.85 1.28 10 A010R R 106.71 1.07 10 A010I I 106.36 1.06 10A010S S 106.34 1.06 10 A010Q Q 106.33 1.06 10 A010P P 105.05 1.05 10A010C C 104.29 1.04 10 A010H H 103.11 1.03 10 A010N N 101.39 1.01 10A010G G 100.93 1.01 10 A010W W 97.00 0.97 10 A010T T 96.44 0.96 10 A010KK 59.66 0.60 10 A010D D 45.09 0.45 10 A010L L 30.80 0.31 10 A010F F 8.410.08 11 S011G G 124.00 1.24 11 S011M M 114.00 1.14 11 S011P P 113.001.13 11 S011C C 112.00 1.12 11 S011F F 112.00 1.12 11 S011V V 104.161.04 11 S011N N 104.00 1.04 11 S011A A 100.00 1.00 11 S011Y Y 96.00 0.9611 S011D D 95.00 0.95 11 S011T T 95.00 0.95 11 S011L L 92.00 0.92 11S011Q Q 92.00 0.92 11 S011I I 90.00 0.90 11 S011W W 88.00 0.88 11 S011KK 66.00 0.66 11 S011R R 43.00 0.43 12 T012A A 142.37 1.42 12 T012G G134.84 1.35 12 T012H H 131.21 1.31 12 T012C C 131.00 1.31 12 T012W W127.05 1.27 12 T012S S 126.74 1.27 12 T012V V 125.13 1.25 12 T012M M123.79 1.24 12 T012P P 120.33 1.20 12 T012I I 117.21 1.17 12 T012Q Q111.02 1.11 12 T012F F 105.34 1.05 12 T012N N 97.46 0.97 12 T012E E82.85 0.83 12 T012K K 76.37 0.76 12 T012R R 51.07 0.51 12 T012D D 50.050.50 12 T012L L 7.07 0.07 13 A013G G 127.43 1.27 13 A013V V 119.50 1.2013 A013S S 106.50 1.07 13 A013Q Q 105.87 1.06 13 A013F F 101.75 1.02 13A013C C 100.86 1.01 13 A013T T 95.70 0.96 13 A013M M 91.53 0.92 13 A013NN 86.10 0.86 13 A013W W 85.59 0.86 13 A013E E 83.94 0.84 13 A013P P72.98 0.73 13 A013D D 50.95 0.51 13 A013R R 27.55 0.28 13 A013H H 7.610.08 13 A013L L 7.22 0.07 14 L014S S 150.94 1.51 14 L014V V 144.29 1.4414 L014A A 141.26 1.41 14 L014F F 129.64 1.30 14 L014W W 125.51 1.26 14L014M M 117.13 1.17 14 L014G G 107.37 1.07 14 L014I I 95.68 0.96 14L014H H 89.97 0.90 14 L014N N 78.82 0.79 14 L014Q Q 67.18 0.67 14 L014YY 62.88 0.63 14 L014K K 41.53 0.42 14 L014E E 40.85 0.41 14 L014R R32.05 0.32 14 L014P P 8.63 0.09 15 L015G G 144.04 1.44 15 L015T T 134.291.34 15 L015M M 128.01 1.28 15 L015C C 125.90 1.26 15 L015V V 119.251.19 15 L015Y Y 118.30 1.18 15 L015W W 116.98 1.17 15 L015A A 109.901.10 15 L015F F 102.58 1.03 15 L015S S 98.62 0.99 15 L015P P 84.79 0.8515 L015Q Q 53.19 0.53 15 L015K K 49.84 0.50 15 L015N N 43.69 0.44 15L015H H 41.81 0.42 15 L015E E 41.21 0.41 15 L015R R 10.80 0.11 16 I016WW 153.84 1.54 16 I016S S 129.21 1.29 16 I016G G 122.70 1.23 16 I016A A116.84 1.17 16 I016C C 109.27 1.09 16 I016V V 103.40 1.03 16 I016Y Y101.35 1.01 16 I016T T 88.85 0.89 16 I016H H 75.02 0.75 16 I016N N 71.580.72 16 I016F F 57.93 0.58 16 I016E E 38.94 0.39 16 I016P P 23.34 0.2316 I016R R 18.70 0.19 16 I016L L 11.62 0.12 17 S017M M 129.94 1.30 17S017A A 129.04 1.29 17 S017R R 126.92 1.27 17 S017L L 118.09 1.18 17S017H H 116.37 1.16 17 S017K K 114.85 1.15 17 S017V V 112.73 1.13 17S017D D 112.49 1.12 17 S017C C 110.18 1.10 17 S017G G 108.87 1.09 17S017E E 108.53 1.09 17 S017Y Y 104.68 1.05 17 S017I I 91.72 0.92 17S017T T 88.65 0.89 17 S017F F 86.66 0.87 17 S017P P 83.37 0.83 18 V018MM 141.00 1.41 18 V018T T 118.00 1.18 18 V018A A 117.48 1.17 18 V018W W112.69 1.13 18 V018E E 110.30 1.10 18 V018S S 104.38 1.04 18 V018R R102.00 1.02 18 V018C C 99.00 0.99 18 V018D D 92.82 0.93 18 V018F F 90.000.90 18 V018 8 86.00 0.86 18 V018P P 80.92 0.81 18 V018H H 76.00 0.76 18V018L L 53.62 0.54 18 V018I I 50.32 0.50 18 V018G G 49.92 0.50 18 V018YY 42.71 0.43 19 A019E E 142.51 1.43 19 A019M M 121.02 1.21 19 A019C C114.96 1.15 19 A019S S 113.37 1.13 19 A019L L 112.60 1.13 19 A019W W109.73 1.10 19 A019V V 109.69 1.10 19 A019G G 108.40 1.08 19 A019T T103.33 1.03 19 A019I I 91.94 0.92 19 A019K K 91.82 0.92 19 A019F F 84.120.84 19 A019R R 81.36 0.81 19 A019P P 80.92 0.81 20 F020N N 123.06 1.2320 F020Q Q 118.20 1.18 20 F020M M 115.31 1.15 20 F020V V 107.59 1.08 20F020S S 107.24 1.07 20 F020T T 104.65 1.05 20 F020L L 100.66 1.01 20F020G G 99.06 0.99 20 F020D D 94.64 0.95 20 F020I I 93.21 0.93 20 F020EE 89.45 0.89 20 F020K K 89.03 0.89 20 F020R R 71.76 0.72 20 F020A A 6.860.07 21 S021T T 98.31 0.98 21 S021E E 90.76 0.91 21 S021Q Q 86.03 0.8621 S021M M 82.73 0.83 21 S021G G 81.75 0.82 21 S021A A 81.13 0.81 21S021W W 80.36 0.80 21 S021V V 79.71 0.80 21 S021R R 79.66 0.80 21 S021KK 77.77 0.78 21 S021L L 72.62 0.73 21 S021N N 71.73 0.72 21 S021I I66.14 0.66 21 S021C C 39.26 0.39 21 S021Y Y 39.16 0.39 21 S021P P 30.330.30 21 S021F F 8.13 0.08 21 S021H H 7.67 0.08 22 S022M M 154.51 1.55 22S022V V 121.71 1.22 22 S022D D 119.67 1.20 22 S022W W 116.41 1.16 22S022G G 115.06 1.15 22 S022T T 109.16 1.09 22 S022R R 108.79 1.09 22S022P P 108.47 1.08 22 S022Y Y 106.89 1.07 22 S022A A 102.30 1.02 22S022L L 97.24 0.97 22 S022F F 95.17 0.95 22 S022E E 93.31 0.93 22 S022HH 92.95 0.93 22 S022C C 89.96 0.90 23 S023K K 128.31 1.28 23 S023G G119.02 1.19 23 S023E E 114.55 1.15 23 S023H H 113.30 1.13 23 S023R R113.12 1.13 23 S023T T 112.61 1.13 23 S023A A 91.93 0.92 23 S023M M91.58 0.92 23 S023P P 91.42 0.91 23 S023V V 88.72 0.89 23 S023C C 86.610.87 23 S023Q Q 83.15 0.83 23 S023L L 77.16 0.77 23 S023I I 74.48 0.7423 S023W W 36.43 0.36 23 S023D D 7.46 0.07 24 I024T T 140.91 1.41 24I024V V 128.01 1.28 24 I024N N 110.91 1.11 24 I024R R 110.89 1.11 24I024L L 105.32 1.05 24 I024H H 102.41 1.02 24 I024P P 101.05 1.01 24I024S S 100.69 1.01 24 I024C C 98.49 0.98 24 I024A A 95.72 0.96 24 I024WW 94.63 0.95 24 I024K K 94.07 0.94 24 I024M M 93.64 0.94 24 I024Y Y 8.750.09 24 I024E E 6.70 0.07 25 A025V V 88.01 0.88 25 A025T T 63.92 0.64 25A025G G 63.07 0.63 25 A025S S 57.55 0.58 25 A025I I 51.29 0.51 25 A025LL 43.81 0.44 25 A025E E 32.21 0.32 25 A025M M 31.37 0.31 25 A025P P29.29 0.29 25 A025K K 27.25 0.27 25 A025N N 25.98 0.26 25 A025H H 23.740.24 25 A025D D 19.49 0.19 25 A025F F 16.89 0.17 25 A025W W 11.14 0.1126 S026Y Y 129.81 1.30 26 S026L L 120.14 1.20 26 S026A A 112.01 1.12 26S026R R 111.71 1.12 26 S026T T 107.06 1.07 26 S026N N 101.40 1.01 26S026K K 99.57 1.00 26 S026M M 94.81 0.95 26 S026D D 93.37 0.93 26 S026GG 90.33 0.90 26 S026W W 87.38 0.87 26 S026V V 82.41 0.82 26 S026H H81.00 0.81 26 S026I I 76.78 0.77 26 S026P P 47.45 0.47 27 A027G G 67.180.67 27 A027S S 44.42 0.44 27 A027K K 40.64 0.41 27 A027C C 39.47 0.3927 A027P P 39.46 0.39 27 A027R R 37.92 0.38 27 A027H H 36.42 0.36 27A027E E 31.42 0.31 27 A027F F 30.21 0.30 27 A027T T 21.37 0.21 27 A027VV 20.42 0.20 27 A027W W 11.82 0.12 27 A027Q Q 11.60 0.12 27 A027L L 4.860.05 28 A028Q Q 109.07 1.09 28 A028T T 107.38 1.07 28 A028S S 83.96 0.8428 A028R R 80.35 0.80 28 A028M M 79.43 0.79 28 A028E E 71.65 0.72 28A028G G 68.05 0.68 28 A028D D 61.77 0.62 28 A028N N 57.89 0.58 28 A028LL 55.70 0.56 28 A028V V 51.81 0.52 28 A028F F 31.30 0.31 28 A028P P27.66 0.28 28 A028W W 24.87 0.25 29 E029T T 133.40 1.33 29 E029Y Y124.28 1.24 29 E029V V 117.64 1.18 29 E029Q Q 115.48 1.15 29 E029K K114.06 1.14 29 E029M M 113.42 1.13 29 E029P P 111.96 1.12 29 E029N N111.04 1.11 29 E029S S 107.45 1.07 29 E029G G 107.33 1.07 29 E029A A107.09 1.07 29 E029C C 87.77 0.88 29 E029L L 85.82 0.86 29 E029I I 80.970.81 29 E029H H 72.39 0.72 29 E029W W 65.30 0.65 29 E029F F 58.62 0.5930 E030Q Q 125.96 1.26 30 E030K K 120.69 1.21 30 E030M M 117.64 1.18 30E030A A 112.55 1.13 30 E030G G 104.25 1.04 30 E030R R 103.70 1.04 30E030S S 103.37 1.03 30 E030H H 103.05 1.03 30 E030N N 100.98 1.01 30E030T T 100.94 1.01 30 E030V V 99.31 0.99 30 E030L L 97.86 0.98 30 E030FF 87.10 0.87 30 E030C C 71.99 0.72 30 E030W W 69.05 0.69 30 E030P P 6.540.07 31 A031R R 123.14 1.23 31 A031G G 118.11 1.18 31 A031S S 110.901.11 31 A031K K 108.32 1.08 31 A031P P 105.73 1.06 31 A031T T 99.73 1.0031 A031M M 96.10 0.96 31 A031V V 95.95 0.96 31 A031L L 75.13 0.75 31A031N N 67.25 0.67 31 A031W W 64.44 0.64 31 A031H H 10.14 0.10 32 K032CC 67.52 0.68 32 K032H H 55.09 0.55 32 K032T T 54.10 0.54 32 K032W W50.65 0.51 32 K032N N 47.14 0.47 32 K032L L 44.25 0.44 32 K032R R 37.800.38 32 K032F F 37.58 0.38 32 K032V V 33.13 0.33 32 K032S S 30.77 0.3132 K032P P 27.44 0.27 32 K032I I 15.39 0.15 32 K032G G 12.63 0.13 32K032Y Y 10.28 0.10 33 E033R R 197.92 1.98 33 E033Q Q 194.17 1.94 33E033G G 146.63 1.47 33 E033H H 132.79 1.33 33 E033N N 129.84 1.30 33E033S S 128.25 1.28 33 E033D D 125.01 1.25 33 E033I I 124.33 1.24 33E033K K 116.99 1.17 33 E033M M 105.94 1.06 33 E033L L 94.42 0.94 33E033T T 89.95 0.90 33 E033Y Y 72.82 0.73 33 E033F F 71.77 0.72 33 E033PP 38.46 0.38 34 K034R R 111.00 1.11 34 K034H H 86.37 0.86 34 K034Q Q64.00 0.64 34 K034N N 56.38 0.56 34 K034P P 55.00 0.55 34 K034T T 55.000.55 34 K034M M 52.00 0.52 34 K034C C 48.00 0.48 34 K034V V 46.00 0.4634 K034L L 35.00 0.35 34 K034G G 34.00 0.34 34 K034D D 27.00 0.27 34K034F F 25.00 0.25 34 K034Y Y 24.00 0.24 34 K034S S 5.00 0.05 35 Y035F F92.05 0.92 35 Y035W W 29.00 0.29 35 Y035V V 23.00 0.23 35 Y035L L 11.960.12 35 Y035C C 11.74 0.12 35 Y035A A 9.31 0.09 35 Y035R R 8.51 0.09 35Y035K K 8.00 0.08 35 Y035S S 7.60 0.08 35 Y035Q Q 7.00 0.07 35 Y035P P6.25 0.06 35 Y035D D 6.15 0.06 35 Y035E E 6.10 0.06 35 Y035N N 6.00 0.0635 Y035T T 6.00 0.06 35 Y035G G 5.85 0.06 36 L036M M 97.00 0.97 36 L036GG 33.00 0.33 36 L036T T 33.00 0.33 36 L036Y Y 25.04 0.25 36 L036C C24.00 0.24 36 L036N N 20.00 0.20 36 L036F F 19.00 0.19 36 L036V V 17.000.17 36 L036W W 9.00 0.09 36 L036R R 8.00 0.08 36 L036A A 7.00 0.07 36L036S S 7.00 0.07 36 L036Q Q 6.21 0.06 36 L036H H 6.08 0.06 36 L036P P5.71 0.06 36 L036D D 3.67 0.04 37 I037V V 59.70 0.60 37 I037H H 22.950.23 37 I037C C 9.56 0.10 37 I037S S 9.48 0.09 37 I037N N 8.54 0.09 37I037A A 8.15 0.08 37 I037F F 7.80 0.08 37 I037P P 7.57 0.08 37 I037L L7.27 0.07 37 I037Y Y 7.00 0.07 37 I037T T 6.86 0.07 37 I037G G 6.86 0.0737 I037W W 6.53 0.07 37 I037D D 6.46 0.06 37 I037Q Q 6.45 0.06 37 I037RR 5.83 0.06 38 G038A A 56.00 0.56 38 G038H H 34.76 0.35 38 G038I I 32.000.32 38 G038S S 31.00 0.31 38 G038V V 31.00 0.31 38 G038T T 24.00 0.2438 G038M M 21.00 0.21 38 G038L L 15.10 0.15 38 G038N N 15.00 0.15 38G038R R 13.00 0.13 38 G038K K 12.55 0.13 38 G038C C 12.00 0.12 38 G038PP 10.00 0.10 38 G038W W 9.87 0.10 38 G038D D 8.00 0.08 38 G038E E 8.000.08 38 G038Y Y 7.09 0.07 39 F039L L 14.25 0.14 39 F039M M 14.10 0.14 39F039W W 10.45 0.10 39 F039Y Y 9.72 0.10 39 F039S S 8.78 0.09 39 F039R R8.71 0.09 39 F039P P 8.06 0.08 39 F039D D 7.82 0.08 39 F039E E 7.79 0.0839 F039V V 7.59 0.08 39 F039C C 7.51 0.08 39 F039A A 6.77 0.07 39 F039KK 6.00 0.06 39 F039H H 5.90 0.06 39 F039G G 5.87 0.06 39 F039N N 5.760.06 39 F039Q Q 5.54 0.06 39 F039T T 5.39 0.05 40 N040V V 126.00 1.26 40N040K K 118.87 1.19 40 N040E E 110.47 1.10 40 N040Q Q 109.00 1.09 40N040H H 107.23 1.07 40 N040Y Y 106.81 1.07 40 N040T T 106.00 1.06 40N040A A 104.00 1.04 40 N040L L 104.00 1.04 40 N040W W 102.81 1.03 40N040F F 102.53 1.03 40 N040R R 102.00 1.02 40 N040C C 98.00 0.98 40N040P P 98.00 0.98 40 N040I I 88.00 0.88 40 N040G G 82.00 0.82 40 N040DD 72.46 0.72 40 N040M M 70.39 0.70 40 N040S S 19.00 0.19 41 E041A A142.97 1.43 41 E041S S 125.74 1.26 41 E041N N 113.99 1.14 41 E041T T113.64 1.14 41 E041H H 108.25 1.08 41 E041R R 104.86 1.05 41 E041G G97.74 0.98 41 E041Y Y 95.99 0.96 41 E041Q Q 92.70 0.93 41 E041F F 63.240.63 41 E041L L 60.52 0.61 41 E041I I 52.81 0.53 41 E041V V 45.47 0.4541 E041W W 32.22 0.32 41 E041P P 9.27 0.09 42 Q042M M 148.09 1.48 42Q042R R 130.84 1.31 42 Q042P P 121.40 1.21 42 Q042W W 113.30 1.13 42Q042S S 95.75 0.96 42 Q042Y Y 86.74 0.87 42 Q042K K 83.66 0.84 42 Q042FF 75.21 0.75 42 Q042H H 74.84 0.75 42 Q042L L 73.83 0.74 42 Q042A A73.23 0.73 42 Q042I I 67.65 0.68 42 Q042E E 58.12 0.58 42 Q042D D 57.230.57 42 Q042T T 57.19 0.57 42 Q042V V 42.92 0.43 42 Q042C C 42.91 0.4342 Q042N N 27.71 0.28 43 E043S S 163.96 1.64 43 E043R R 124.40 1.24 43E043D D 120.22 1.20 43 E043H H 105.43 1.05 43 E043A A 104.90 1.05 43E043L L 95.08 0.95 43 E043N N 91.28 0.91 43 E043G G 90.19 0.90 43 E043FF 69.79 0.70 43 E043Y Y 66.91 0.67 43 E043C C 66.68 0.67 43 E043W W64.35 0.64 43 E043V V 53.37 0.53 43 E043P P 42.96 0.43 43 E043M M 8.820.09 43 E043T T 5.27 0.05 44 A044D D 136.49 1.36 44 A044Q Q 113.72 1.1444 A044E E 108.45 1.08 44 A044S S 102.67 1.03 44 A044N N 90.51 0.91 44A044R R 82.63 0.83 44 A044G G 82.29 0.82 44 A044C C 80.18 0.80 44 A044LL 79.49 0.79 44 A044H H 69.28 0.69 44 A044V V 50.75 0.51 44 A044Y Y44.57 0.45 44 A044T T 42.35 0.42 44 A044I I 29.22 0.29 44 A044P P 16.750.17 44 A044F F 8.08 0.08 45 V045L L 128.46 1.28 45 V045A A 122.17 1.2245 V045C C 117.75 1.18 45 V045R R 89.35 0.89 45 V045T T 83.00 0.83 45V045I I 81.29 0.81 45 V045Q Q 69.47 0.69 45 V045F F 42.91 0.43 45 V045YY 38.60 0.39 45 V045S S 31.46 0.31 45 V045D D 30.61 0.31 45 V045H H15.52 0.16 45 V045P P 13.16 0.13 45 V045N N 8.45 0.08 46 S046H H 180.171.80 46 S046R R 163.56 1.64 46 S046T T 145.04 1.45 46 S046A A 134.771.35 46 S046Q Q 133.68 1.34 46 S046Y Y 126.03 1.26 46 S046W W 108.601.09 46 S046V V 99.68 1.00 46 S046L L 95.29 0.95 46 S046G G 94.57 0.9546 S046D D 94.34 0.94 46 S046E E 80.44 0.80 46 S046F F 68.99 0.69 46S046P P 46.63 0.47 47 E047D D 161.68 1.62 47 E047R R 127.01 1.27 47E047K K 105.12 1.05 47 E047Q Q 103.73 1.04 47 E047H H 102.20 1.02 47E047A A 99.30 0.99 47 E047Y Y 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0.26 81 L081T T 15.280.15 81 L081F F 11.66 0.12 81 L081W W 9.72 0.10 81 L081Y Y 8.23 0.08 81L081S S 7.53 0.08 81 L081P P 7.23 0.07 81 L081D D 6.93 0.07 81 L081G G6.62 0.07 81 L081E E 6.60 0.07 81 L081H H 5.90 0.06 81 L081R R 5.68 0.0682 S082L L 129.13 1.29 82 S082Q Q 128.15 1.28 82 S082V V 103.94 1.04 82S082H H 97.35 0.97 82 S082Y Y 91.12 0.91 82 S082W W 88.77 0.89 82 S082AA 87.22 0.87 82 S082E E 85.81 0.86 82 S082F F 81.39 0.81 82 S082C C79.80 0.80 82 S082N N 61.37 0.61 82 S082R R 58.97 0.59 82 S082G G 54.280.54 82 S082T T 54.07 0.54 82 S082D D 18.02 0.18 82 S082P P 6.19 0.06 83V083N N 109.58 1.10 83 V083Y Y 102.00 1.02 83 V083L L 97.61 0.98 83V083G G 42.23 0.42 83 V083H H 33.13 0.33 83 V083K K 11.63 0.12 83 V083CC 10.69 0.11 83 V083T T 7.51 0.08 83 V083Q Q 7.00 0.07 83 V083D D 6.610.07 83 V083I I 6.32 0.06 83 V083M M 6.20 0.06 83 V083R R 6.04 0.06 83V083E E 5.77 0.06 83 V083P P 5.67 0.06 83 V083S S 4.97 0.05 83 V083W W4.69 0.05 83 V083F F 4.62 0.05 83 V083A A 4.61 0.05 84 E084L L 153.831.54 84 E084K K 150.95 1.51 84 E084V V 148.36 1.48 84 E084M M 139.821.40 84 E084S S 135.46 1.35 84 E084A A 127.39 1.27 84 E084G G 124.121.24 84 E084T T 123.30 1.23 84 E084R R 116.66 1.17 84 E084F F 111.031.11 84 E084Q Q 101.46 1.01 84 E084N N 100.11 1.00 84 E084W W 98.81 0.9984 E084I I 98.06 0.98 84 E084C C 95.71 0.96 84 E084Y Y 89.76 0.90 84E084H H 75.48 0.75 84 E084D D 50.64 0.51 84 E084P P 7.96 0.08 85 L085F F100.71 1.01 85 L085V V 87.45 0.87 85 L085A A 66.38 0.66 85 L085G G 41.130.41 85 L085T T 27.10 0.27 85 L085Q Q 12.11 0.12 85 L085N N 11.63 0.1285 L085R R 10.96 0.11 85 L085W W 8.95 0.09 85 L085H H 7.59 0.08 85 L085EE 6.71 0.07 85 L085K K 6.71 0.07 86 S086R R 133.45 1.33 86 S086D D124.54 1.25 86 S086K K 120.11 1.20 86 S086N N 118.58 1.19 86 S086A A114.90 1.15 86 S086C C 94.83 0.95 86 S086L L 80.83 0.81 86 S086M M 77.930.78 86 S086Q Q 77.62 0.78 86 S086E E 61.08 0.61 86 S086I I 45.96 0.4686 S086G G 43.12 0.43 86 S086P P 40.52 0.41 87 P087K K 89.04 0.89 87P087R R 62.86 0.63 87 P087T T 58.88 0.59 87 P087H H 58.22 0.58 87 P087SS 56.23 0.56 87 P087N N 51.90 0.52 87 P087V V 47.19 0.47 87 P087Q Q46.90 0.47 87 P087I I 38.44 0.38 87 P087G G 35.49 0.35 87 P087M M 32.830.33 87 P087D D 27.06 0.27 87 P087L L 25.98 0.26 87 P087W W 15.17 0.1588 E088P P 128.01 1.28 88 E088D D 103.64 1.04 88 E088G G 98.73 0.99 88E088I I 76.72 0.77 88 E088Y Y 73.31 0.73 88 E088R R 71.94 0.72 88 E088TT 71.19 0.71 88 E088V V 69.78 0.70 88 E088W W 59.58 0.60 88 E088S S57.12 0.57 88 E088Q Q 45.20 0.45 88 E088N N 32.31 0.32 88 E088H H 31.970.32 88 E088L L 28.70 0.29 88 E088K K 24.60 0.25 89 D089K K 110.11 1.1089 D089H H 101.55 1.02 89 D089A A 97.39 0.97 89 D089N N 88.63 0.89 89D089C C 86.69 0.87 89 D089S S 68.40 0.68 89 D089M M 60.16 0.60 89 D089TT 57.25 0.57 89 D089R R 56.85 0.57 89 D089F F 56.61 0.57 89 D089Q Q56.61 0.57 89 D089G G 50.16 0.50 89 D089V V 49.23 0.49 89 D089E E 38.220.38 89 D089L L 10.85 0.11 89 D089P P 7.52 0.08 90 V090K K 93.59 0.94 90V090A A 77.43 0.77 90 V090L L 74.46 0.74 90 V090R R 70.69 0.71 90 V090II 68.15 0.68 90 V090C C 56.98 0.57 90 V090T T 51.24 0.51 90 V090S S23.79 0.24 90 V090M M 23.60 0.24 90 V090H H 21.17 0.21 90 V090Y Y 14.440.14 90 V090F F 13.27 0.13 90 V090W W 11.98 0.12 90 V090G G 10.52 0.1190 V090P P 7.80 0.08 90 V090N N 7.52 0.08 90 V090D D 7.09 0.07 91 D091EE 148.69 1.49 91 D091P P 144.01 1.44 91 D091A A 142.94 1.43 91 D091T T136.68 1.37 91 D091N N 124.34 1.24 91 D091K K 118.47 1.18 91 D091W W108.37 1.08 91 D091R R 107.58 1.08 91 D091F F 104.13 1.04 91 D091M M98.03 0.98 91 D091C C 97.38 0.97 91 D091V V 87.03 0.87 91 D091L L 81.600.82 91 D091G G 18.66 0.19 91 D091S S 5.81 0.06 92 A092R R 139.58 1.4092 A092K K 135.47 1.35 92 A092T T 134.11 1.34 92 A092M M 132.58 1.33 92A092V V 130.35 1.30 92 A092Q Q 121.93 1.22 92 A092I I 119.20 1.19 92A092L L 117.04 1.17 92 A092S S 105.71 1.06 92 A092E E 97.94 0.98 92A092C C 89.25 0.89 92 A092H H 88.18 0.88 92 A092Y Y 84.08 0.84 92 A092DD 83.51 0.84 92 A092F F 76.69 0.77 92 A092W W 70.16 0.70 92 A092P P 8.530.09 92 A092G G 7.42 0.07 93 L093F F 60.69 0.61 93 L093I I 53.14 0.53 93L093M M 51.15 0.51 93 L093V V 44.68 0.45 93 L093Y Y 12.17 0.12 93 L093WW 11.12 0.11 93 L093A A 11.07 0.11 93 L093T T 9.55 0.10 93 L093N N 7.930.08 93 L093R R 7.73 0.08 93 L093Q Q 7.40 0.07 93 L093K K 7.36 0.07 93L093G G 7.31 0.07 93 L093E E 7.18 0.07 93 L093P P 7.17 0.07 93 L093H H6.92 0.07 93 L093S S 6.62 0.07 93 L093C C 6.43 0.06 94 E094A A 130.001.30 94 E094M M 127.00 1.27 94 E094T T 125.00 1.25 94 E094R R 115.001.15 94 E094S S 111.00 1.11 94 E094L L 103.00 1.03 94 E094F F 96.41 0.9694 E094V V 94.00 0.94 94 E094C C 88.52 0.89 94 E094N N 86.00 0.86 94E094D D 76.35 0.76 94 E094G G 71.00 0.71 94 E094P P 15.00 0.15 94 E094II 6.00 0.06 94 E094K K 6.00 0.06 95 L095E E 144.50 1.44 95 L095R R143.47 1.43 95 L095K K 132.53 1.33 95 L095S S 127.82 1.28 95 L095C C126.67 1.27 95 L095A A 123.21 1.23 95 L095T T 119.38 1.19 95 L095D D112.59 1.13 95 L095G G 107.90 1.08 95 L095V V 101.75 1.02 95 L095H H101.25 1.01 95 L095Y Y 72.15 0.72 95 L095I I 66.43 0.66 95 L095F F 55.600.56 95 L095W W 54.88 0.55 95 L095M M 49.85 0.50 95 L095P P 43.73 0.4495 L095N N 7.87 0.08 96 D096Y Y 154.21 1.54 96 D096H H 154.17 1.54 96D096C C 131.97 1.32 96 D096S S 125.64 1.26 96 D096L L 122.29 1.22 96D096E E 115.11 1.15 96 D096I I 111.95 1.12 96 D096W W 111.27 1.11 96D096V V 103.05 1.03 96 D096T T 91.78 0.92 96 D096F F 81.46 0.81 96 D096GG 66.73 0.67 96 D096R R 54.25 0.54 96 D096P P 30.89 0.31 96 D096K K24.76 0.25 96 D096M M 7.93 0.08 96 D096N N 7.71 0.08 97 P097E E 73.240.73 97 P097S S 66.40 0.66 97 P097D D 63.49 0.63 97 P097A A 58.83 0.5997 P097K K 57.21 0.57 97 P097Q Q 54.30 0.54 97 P097N N 47.24 0.47 97P097G G 46.89 0.47 97 P097T T 45.56 0.46 97 P097R R 45.09 0.45 97 P097MM 33.57 0.34 97 P097C C 29.31 0.29 97 P097V V 22.34 0.22 97 P097Y Y21.56 0.22 97 P097F F 20.98 0.21 97 P097L L 19.17 0.19 97 P097I I 18.760.19 97 P097W W 15.10 0.15 98 A098R R 132.47 1.32 98 A098K K 131.64 1.3298 A098D D 102.97 1.03 98 A098L L 102.06 1.02 98 A098T T 91.56 0.92 98A098G G 89.92 0.90 98 A098V V 70.86 0.71 98 A098M M 48.09 0.48 98 A098FF 40.13 0.40 98 A098E E 36.13 0.36 99 I099V V 77.11 0.77 99 I099F F13.83 0.14 99 I099L L 13.75 0.14 99 I099T T 10.36 0.10 99 I099P P 7.860.08 99 I099Q Q 7.27 0.07 99 I099E E 7.21 0.07 99 I099K K 6.92 0.07 99I099A A 6.85 0.07 99 I099H H 6.53 0.07 99 I099D D 6.44 0.06 99 I099S S6.42 0.06 99 I099G G 6.26 0.06 99 I099W W 6.22 0.06 99 I099R R 5.85 0.0699 I099Y Y 5.59 0.06 100 S100A A 103.00 1.03 100 S100V V 97.27 0.97 100S100R R 91.59 0.92 100 S100E E 87.00 0.87 100 S100T T 81.00 0.81 100S100C C 75.00 0.75 100 S100L L 61.00 0.61 100 S100H H 59.00 0.59 100S100I I 53.00 0.53 100 S100W W 43.04 0.43 100 S100G G 35.00 0.35 100S100Q Q 21.00 0.21 100 S100F F 13.00 0.13 100 S100M M 7.00 0.07 101Y101F F 79.08 0.79 101 Y101H H 34.40 0.34 101 Y101R R 8.50 0.09 101Y101T T 7.52 0.08 101 Y101C C 7.50 0.08 101 Y101Q Q 7.46 0.07 101 Y101SS 7.43 0.07 101 Y101L L 7.42 0.07 101 Y101N N 7.33 0.07 101 Y101G G 7.030.07 101 Y101E E 6.76 0.07 101 Y101P P 6.39 0.06 102 I102L L 32.85 0.33102 I102T T 12.31 0.12 102 I102A A 11.19 0.11 102 I102F F 7.57 0.08 102I102P P 7.53 0.08 102 I102G G 6.21 0.06 102 I102K K 5.78 0.06 102 I102SS 5.77 0.06 102 I102R R 5.60 0.06 102 I102Y Y 5.40 0.05 102 I102H H 5.150.05 102 I102E E 5.10 0.05 102 I102Q Q 4.91 0.05 103 E103T T 113.38 1.13103 E103P P 111.36 1.11 103 E103S S 86.19 0.86 103 E103G G 81.52 0.82103 E103D D 56.43 0.56 103 E103H H 33.35 0.33 103 E103Q Q 10.36 0.10 103E103I I 8.00 0.08 103 E103A A 7.62 0.08 103 E103K K 7.55 0.08 103 E103LL 7.25 0.07 103 E103W W 7.22 0.07 103 E103C C 7.10 0.07 103 E103F F 6.980.07 103 E103V V 6.88 0.07 103 E103R R 6.04 0.06 104 E104P P 124.98 1.25104 E104Q Q 98.83 0.99 104 E104L L 95.33 0.95 104 E104Y Y 91.66 0.92 104E104H H 87.15 0.87 104 E104S S 81.74 0.82 104 E104A A 76.42 0.76 104E104F F 71.58 0.72 104 E104T T 69.53 0.70 104 E104C C 64.06 0.64 104E104V V 57.67 0.58 104 E104N N 48.45 0.48 104 E104W W 43.19 0.43 104E104G G 33.84 0.34 105 D105M M 25.47 0.25 105 D105T T 16.88 0.17 105D105E E 14.77 0.15 105 D105P P 7.10 0.07 105 D105C C 6.97 0.07 105 D105GG 6.51 0.07 105 D105S S 6.19 0.06 105 D105F F 5.83 0.06 105 D105L L 5.710.06 105 D105W W 5.66 0.06 105 D105Y Y 5.36 0.05 106 A106F F 108.54 1.09106 A106K K 106.67 1.07 106 A106V V 106.21 1.06 106 A106L L 105.12 1.05106 A106M M 102.59 1.03 106 A106C C 95.21 0.95 106 A106S S 94.82 0.95106 A106I I 88.65 0.89 106 A106E E 84.86 0.85 106 A106N N 80.65 0.81 106A106G G 66.53 0.67 106 A106D D 44.78 0.45 107 E107Q Q 112.74 1.13 107E107P P 102.05 1.02 107 E107V V 99.68 1.00 107 E107S S 99.06 0.99 107E107T T 93.40 0.93 107 E107H H 89.38 0.89 107 E107R R 87.40 0.87 107E107L L 82.19 0.82 107 E107G G 65.90 0.66 107 E107C C 34.35 0.34 107E107D D 6.32 0.06 107 E107Y Y 5.97 0.06 107 E107I I 5.76 0.06 108 V108II 96.87 0.97 108 V108A A 81.56 0.82 108 V108L L 73.95 0.74 108 V108C C73.59 0.74 108 V108T T 54.76 0.55 108 V108G G 34.72 0.35 108 V108E E13.15 0.13 108 V108S S 6.94 0.07 108 V108H H 6.42 0.06 108 V108P P 6.030.06 108 V108Q Q 6.03 0.06 108 V108R R 5.94 0.06 109 T109Y Y 151.13 1.51109 T109E E 140.77 1.41 109 T109G G 135.65 1.36 109 T109N N 135.23 1.35109 T109S S 125.47 1.25 109 T109M M 123.65 1.24 109 T109R R 121.74 1.22109 T109Q Q 105.15 1.05 109 T109W W 102.35 1.02 109 T109K K 98.87 0.99109 T109L L 95.89 0.96 109 T109A A 31.57 0.32 110 T110V V 117.34 1.17110 T110L L 113.17 1.13 110 T110P P 112.26 1.12 110 T110M M 110.23 1.10110 T110C C 98.41 0.98 110 T110K K 97.01 0.97 110 T110E E 80.71 0.81 110T110G G 80.05 0.80 110 T110R R 79.50 0.80 110 T110N N 74.55 0.75 110T110Q Q 57.23 0.57 110 T110S S 5.27 0.05 110 T110W W 4.82 0.05 111 M111NN 145.51 1.46 111 M111T T 138.47 1.38 111 M111Q Q 134.85 1.35 111 M111LL 134.45 1.34 111 M111W W 132.12 1.32 111 M111E E 130.67 1.31 111 M111CC 113.25 1.13 111 M111G G 101.14 1.01 111 M111I I 100.83 1.01 111 M111VV 93.69 0.94 111 M111R R 74.46 0.74 111 M111K K 70.17 0.70 111 M111P P31.70 0.32 *“POSITION” refers to the amino acid position as numbered inthe full-length V049 precursor protease of SEQ ID NO: 13. Positions 1-27refer to amino acids at positions in the signal peptide portion of theV049 precursor enzyme, and positions 28-111 refer to the amino acids atpositions in the pro region of the V049 precursor enzyme. #ACTIVITYRATIO is the Activity of the mature protease processed from a modifiedprotease divided by the activity of the mature protease processed froman unmodified precursor protease.

Example 4

In this example, the pro region of the subtilisin from Bacillus lentus,called GG36 (SEQ ID NO:244), was mutagenized at position 33 and position59 using the same experimental protocol described in the examples above.The precursor polynucleotide sequence (SEQ ID NO:240) was mutated, aconstruct was made, and the modifications were tested for increasing theprotease expression as described above.

The results showed that amino acid substitutions E33G, E33Q and E33V allincreased the production of the wild-type mature GG36 protease by atleast 50% above that of the unmodified precursor. The mutation H32Kincreased the production of the wild-type mature protease by 20%.

These results show that mutations in the pro region of a full-lengthprotease can improve the expression not only of variant precursorproteins, but also of naturally-occurring enzymes.

We claim:
 1. An isolated modified polynucleotide encoding a modifiedfull-length protease, wherein the portion of said modifiedpolynucleotide that encodes the pro region of said full-length proteasecomprises a mutation encoding a substitution at least at one amino acidposition chosen from positions equivalent to amino acid positions 28-108and 109 of SEQ ID NO:5, SEQ ID NO:13 or SEQ ID NO:244.
 2. The isolatedmodified polynucleotide of claim 1, wherein said modified full-lengthprotease is a serine protease.
 3. The isolated modified polynucleotideof claim 1, wherein said modified full-length protease is an alkalineserine protease derived from a wild-type or variant precursor alkalineserine protease.
 4. The isolated modified polynucleotide of claim 3,wherein said precursor alkaline serine protease is a B. clausii or a B.lentus alkaline serine protease.
 5. The isolated modified polynucleotideof claim 1, wherein said isolated modified polynucleotide comprises thepolynucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:9 or SEQ IDNO:240.
 6. The isolated modified polynucleotide of claim 1, wherein saidportion of said modified polynucleotide that encodes the pro regioncomprises a mutation encoding a substitution at least at one amino acidposition chosen from E33, E43, A44, E47, V49, E57, A59, E63, E70, E74,E84, and E88 of SEQ ID NO:5, 13, or
 244. 7. The isolated polynucleotideof claim 6, wherein said substitution of E33 is chosen from E33D, E33I,E33S, E33N, E33K, E33H, E33Q and E33R.
 8. A vector comprising themodified polynucleotide of claim
 1. 9. A host cell transformed with thevector of claim
 8. 10. The host cell of claim 9, wherein said host cellis a microorganism.
 11. The host cell of claim 9, wherein said host cellis a microorganism chosen from the group consisting of Bacillus sp.,Streptomyces sp., Escherichia sp. and Aspergillus sp.
 12. The host cellof claim 9, wherein said host cell is a B. subtilis cell.
 13. A proteaseproduced by the host cell of claim
 9. 14. A method for producing aprotease in a microorganism comprising culturing the host cell of claim9 under suitable conditions to allow the production of said modifiedfull-length protease by said host cell.
 15. The method of claim 14,wherein the mature region of said modified full-length protease producedby said host cell is recovered.
 16. The method of claim 14, wherein saidmature region is at least about 70% identical to SEQ ID NOS:8, 16 or247.
 17. The method of claim 14, wherein said at least one amino acidsubstitution is chosen from E33D, E331, E33S, E33N, E33K, E33H, E33Q,E33R, E57F, E57W, E57K, E57R, E57D, E57M, E57C, E57Q, E57S, E57H andE57N.
 18. The method of claim 14, wherein said host cell is chosen fromthe group consisting of B. licheniformis, B. lentus, B. subtilis, B.amyloliquefaciens, B. brevis, B. stearothermophilus, B. clausii, B.alkalophilus, B. halodurans, B. coagulans, B. circulans, B. pumilus, andB. thuringiensis.
 19. The method of claim 14, wherein said host cell isa B. subtilis host cell.
 20. The method of claim 14, wherein saidmodified full-length protease exhibits a ratio of production of at least1.